Pharmaceutical compositions containing substituted polycyclic pyridone derivatives and prodrug thereof

ABSTRACT

The present invention provides a pharmaceutical composition containing the following compound having antiviral action:wherein each of the symbols is defined in the specification.

TECHNICAL FIELD

This invention relates to substituted polycyclic pyridone derivativeshaving cap-dependent endonuclease inhibitory activity, prodrugs thereof,and pharmaceutical compositions including thereof.

BACKGROUND ART

Influenza is an acute respiratory infectious disease caused by infectionwith an influenza virus. In Japan, millions of influenza-like patientsare reported every winter, and influenza is accompanied with highmorbidity and mortality. Influenza is a particularly important diseasein a high risk population such as baby and elderly, a complication ratewith pneumonia is high in elderly, and death with influenza is occupiedwith elderly in many cases.

As anti-influenza drugs, Symmetrel (trade name: Amantadine) andFlumadine (trade name: Rimantadine) which inhibit the denucleationprocess of a virus, and Oseltamivir (trade name: Tamiflu) and Zanamivir(trade name: Relenza) which are neuraminidase inhibitors suppressingvirus budding and release from a cell are known. However, since problemsof appearances of resistant strains and side effects, and worldwideepidemic of a new-type influenza virus having high pathogenicity andmortality are feared, development of an anti-influenza drug having anovel mechanism has been desired.

Since a cap-dependent endonuclease which is an influenza virus-derivedenzyme is essential for virus proliferation, and has the virus-specificenzymatic activity which is not possessed by a host, it is believed thatthe endonuclease is suitable for a target of an anti-influenza drug. Thecap-dependent endonuclease of an influenza virus has a host mRNAprecursor as a substrate, and has the endonuclease activity of producinga fragment of 9 to 13 bases including a cap structure (not including thenumber of bases of the cap structure). This fragment functions as aprimer of a virus RNA polymerase, and is used in synthesizing mRNAencoding a virus protein. That is, it is believed that a substance whichinhibits the cap-dependent endonuclease inhibits synthesis of a virusprotein by inhibiting synthesis of virus mRNA and, as a result, inhibitsvirus proliferation.

As the substance which inhibits the cap-dependent endonuclease,flutimide (Patent Document 1 and Non-Patent Documents 1 and 2),4-substituted 2,4-dioxobutanoic acid (Patent Document 2 and Non-PatentDocuments 3 and 4), the compounds described in Patent Documents 3 to 12and the like have been reported, but they have not yet led to clinicaluse as anti-influenza drugs. Patent Documents 9 and 12 describecompounds having a similar structure to that used in the presentinvention, but does not describe the compounds used in the presentinvention. Also, Patent Documents 13 to 15 describe compounds having asimilar structure to that used in the present invention as a compoundhaving integrase inhibitory activity, however, the documents do notdescribe cap-dependent endonuclease. In addition, Patent Document 16 and17 describes an invention relating to compounds having a similarstructure to that used in the present invention as a compound havingcap-dependent endonuclease inhibitory activity, which has been filed bythe applicants, but does not describe the compounds used in the presentinvention.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: GB2280435-   Patent Document 2: U.S. Pat. No. 5,475,109-   Patent Document 3: US20130090300-   Patent Document 4: WO2013/057251-   Patent Document 5: WO2013/174930-   Patent Document 6: WO2014/023691-   Patent Document 7: WO2014/043252-   Patent Document 8: WO2014/074926-   Patent Document 9: WO2014/108406-   Patent Document 10: WO2014/108407-   Patent Document 11: WO2014/108408-   Patent Document 12: WO2015/038655-   Patent Document 13: WO2005/016927-   Patent Document 14: WO2006/066414-   Patent Document 15: WO2007/049675-   Patent Document 16: WO2010/147068-   Patent Document 17: WO2012/039414

Non-Patent Documents

-   Non-Patent Document 1: Tetrahedron Lett 1995, 36(12), 2005-   Non-Patent Document 2: Tetrahedron Lett 1995, 36(12), 2009-   Non-Patent Document 3: Antimicrobial Agents And Chemotherapy,    December 1994, p. 2827-2837-   Non-Patent Document 4: Antimicrobial Agents And Chemotherapy, May    1996, p. 1304-1307

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide pharmaceuticalcomposition containing compounds having antiviral activities, especiallyinhibiting growth activity of influenza virus. Another object of thepresent invention is to provide a pharmaceutical composition containinga prodrug prepared from compounds used for in vivo administration (forexample, oral administration), being efficiently absorbed into the bodyafter administration and showing high pharmacological effect. Yetanother object is to provide a pharmaceutical composition withshortening the time to alleviation of influenza symptoms.

Means for Solving the Problems

The present invention provides inventions shown below.

(1) A pharmaceutical composition comprising a compound represented byformula (I) or its pharmaceutically acceptable salt:

whereinP is hydrogen or a group to form a prodrug;A¹ is CR^(1A)R^(1B), S or O;A² is CR^(2A)R^(2B), S or O;A³ is CR^(3A)R^(3B), S or O;A⁴ is each independently CR^(4A)R^(4B), S or O;the number of hetero atoms among atoms constituting the ring whichconsists of A¹, A², A³, A⁴, nitrogen atom adjacent to A¹ and carbon atomadjacent to A⁴ is 1 or 2;R^(1A) and R^(1B) are each independently hydrogen, halogen, alkyl,haloalkyl, alkyloxy or phenyl;R^(2A) and R^(2B) are each independently hydrogen, halogen, alkyl,haloalkyl, alkyloxy or phenyl;R^(3A) and R^(3B) are each independently hydrogen, halogen, alkyl,haloalkyl, alkyloxy or phenyl;R^(4A) and R^(4B) are each independently hydrogen, halogen, alkyl,haloalkyl, alkyloxy or phenyl;R^(3A) and R^(3B) may be taken together with an adjacent carbon atom toform non-aromatic carbocycle or non-aromatic heterocycle;R¹ is fluorine;m is any integer of 1 to 2; andn is any integer of 1 to 2.(2) The pharmaceutical composition according to (1), comprising thecompound or its pharmaceutically acceptable salt, wherein the grouprepresented by formula:

wherein each definition has the same meanings as described (1)is a group represented by formula:

wherein R², R³, R⁴ and R⁵ are each independently hydrogen or fluorine;the number of fluorine atoms of R², R³, R⁴ and R⁵ is 1 or 2.(3) The pharmaceutical composition according to (1), comprising thecompound or its pharmaceutically acceptable salt, wherein the grouprepresented by formula:

wherein each definition has the same meanings as described (1)is a group represented by formula:

(4) The pharmaceutical composition according to any one of (1) to (3),comprising the compound or its pharmaceutically acceptable salt, whereinthe group represented by formula:

wherein each definition has the same meanings as described (1)is a group represented by formula:

wherein each definition has the same meanings as described (1).(5) The pharmaceutical composition according to (1), comprising thecompound represented by the following formula or its pharmaceuticallyacceptable salt:

wherein each definition has the same meanings as described (1).(6) The pharmaceutical composition according to (1), comprising thecompound represented by the following formula or its pharmaceuticallyacceptable salt:

wherein each definition has the same meanings as described (1).(7) The pharmaceutical composition according to (1), comprising thecompound represented by the following formula or its pharmaceuticallyacceptable salt:

wherein each definition has the same meanings as described (1).(8) The pharmaceutical composition according to (1), comprising thecompound represented by the following formula or its pharmaceuticallyacceptable salt:

wherein each definition has the same meanings as described (1).(9) The pharmaceutical composition according to (1), comprising thecompound represented by the following formula or its pharmaceuticallyacceptable salt:

wherein each definition has the same meanings as described (1).(10) The pharmaceutical composition according to (1), comprising thecompound represented by the following formula or its pharmaceuticallyacceptable salt:

wherein each definition has the same meaning as described (1).(11) A pharmaceutical composition comprising the compound represented bythe following formula or its pharmaceutically acceptable salt:

wherein each definition has the same meaning as described (1).(12) The pharmaceutical composition according to any one of (1) to (11),comprising the compound or its pharmaceutically acceptable salt,wherein the group to form a prodrug is a group selected from thefollowing formula a) to ac):—C(═O)—^(PRO),  a)—C(═O)—P^(R1),  b)—C(═O)-L-P^(R1),  c)—C(═O)-L-O—P^(R1),  d)—C(═O)-L-O-L-O—P^(R1),  e)—C(═O)L-O—C(═O)—P^(R1),  f)—C(═O)—O—P^(R2),  g)—C(═O)—N(—K)(^(PR2)),  h)—C(═O)—O-L-O—^(PR2),  i)—C(P^(R3))₂—O—P^(R4),  j)—C(P^(R3))₂—O-L-O—P^(R4),  k)—C(P^(R3))₂—O—C(═O)—P^(R4),  l)—C(P^(R3))₂—O—C(═O)—O—P^(R4),  m)—C(P^(R3))₂—O—C(═O)—N(—K)—P^(R4),  n)—C(P^(R3))₂—O—C(═O)—O-L-O—P^(R4),  o)—C(P^(R3))₂—O—C(═O)—O-L-N(P^(R4))₂,  p)—C(P^(R3))₂—O—C(═O)—N(—K)-L-O—P^(R4),  q)—C(P^(R3))₂—O—C(═O)—N(—K)-L-N(P^(R4))₂,  r)—C(P^(R3))₂—O—C(═O)—O-L-O-L-O—P^(R4),  s)—C(P^(R3))₂—O—C(═O)—O-L-N(—K)—C(═O)—P^(R4),  t)—C(P^(R3))₂—O—P(═O)(—P^(R5))₂,  u)—C(P^(R3))₂—P^(R6),  v)—C(═N⁺(P^(R7))₂)(—N(P^(R7))₂),  w)—C(^(PR3))₂—C(P^(R3))₂—C(═O)—O—P^(R2),  x)—C(P^(R3))₂—N(—K)—C(═O)—O—P^(R2),  y)—P(═O)(—P^(R8))(—P^(R8)),  z)—S(═O)₂—P^(R10),  aa)—P^(R11), and  ab)—C(P^(R3))₂—C(P^(R3))₂—O—P^(R2),  ac)wherein L is straight or branched alkylene, or straight or branchedalkenylene;K is hydrogen, or alkyl optionally substituted by substituent group A;P^(R0) is alkyl optionally substituted by substituent group A, oralkenyl optionally substituted by substituent group A;P^(R1) is carbocyclyl group optionally substituted by substituent groupA, heterocyclyl group optionally substituted by substituent group A,alkylamino optionally substituted by substituent group A, oralkylsulfanyl optionally substituted by substituent group A;P^(R2) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A,heterocyclyl group optionally substituted by substituent group A,carbocyclyl alkyl optionally substituted by substituent group A,heterocyclylalkyl optionally substituted by substituent group A ortrialkylsilyl;P^(R3) is each independently hydrogen or alkyl;P^(R4) is each independently alkyl optionally substituted by substituentgroup A, carbocyclyl group optionally substituted by substituent groupA, heterocyclyl group optionally substituted by substituent group A,alkylamino optionally substituted by substituent group A,carbocyclylalkyl optionally substituted by substituent group A,heterocyclylalkyl optionally substituted by substituent group A, ortrialkylsilyl;P^(R5) is each independently hydroxy or OBn;P^(R6) is carbocyclyl group optionally substituted by substituent groupA, or heterocyclyl group optionally substituted by substituent group A;P^(R7) is each independently alkyl optionally substituted by substituentgroup A;P^(R8) is alkyloxy optionally substituted by substituent group A;P^(R9) is alkyloxy optionally substituted by substituent group A,alkylamino optionally substituted by substituent group A, carbocyclyloxyoptionally substituted by substituent group A, heterocyclyloxyoptionally substituted by substituent group A, carbocyclylaminooptionally substituted by substituent group A or heterocyclylaminooptionally substituted by substituent group A;P^(R8) and P^(R9) may be taken together with an adjacent phosphorus atomto form heterocycle optionally substituted by substituent group A;P^(R10) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A,heterocyclyl group optionally substituted by substituent group A,carbocyclylalkyl optionally substituted by substituent group A orheterocyclylalkyl optionally substituted by substituent group A; andP^(R11) is alkyl optionally substituted by substituent group A, alkenyloptionally substituted by substituent group A, carbocyclyl groupoptionally substituted by substituent group A, or heterocyclyl groupoptionally substituted by substituent group A;Substituent group A; oxo, alkyl, hydroxyalkyl, amino, alkylamino,carbocyclyl group, heterocyclyl group, carbocyclylalkyl, alkylcarbonyl,halogen, hydroxy, carboxy, alkylcarbonylamino, alkylcarbonylaminoalkyl,alkylcarbonyloxy, alkyloxycarbonyl, aryloxycarbonylalkyl,alkyloxycarbonyloxy, alkylaminocarbonyloxy, alkylaminoalkyl, alkyloxy,cyano, nitro, azido, alkylsulfonyl, trialkylsilyl and phospho.(13) The pharmaceutical composition according to (12), comprising thecompound or its pharmaceutically acceptable salt,wherein the group to form a prodrug is a group selected from thefollowing formula:—C(═O)—P^(R0),  a)—C(═O)—P^(R1),  b)—C(═O)—O—P^(R2),  g)—C(═O)—N(—K)(P^(R2)),  h)—C(═O)—O-L-O—P^(R2),  i)—C(P^(R3))₂—O—C(═O)—P^(R4),  l)—C(P^(R3))₂—O—C(═O)—O—P^(R4),  m)—C(P^(R3))₂—OC(═O)—O-L-O—P^(R4),  o)—C(P^(R3))₂—P^(R6),  v)—C(P^(R3))₂—C(P^(R3))₂—C(═O)—O—P^(R2),  x)—C(P^(R3))₂—N(—K)—C(═O)—O—P^(R2), and  y)—P(═O)(—P^(R8))(—P^(R9)),  z)wherein L is straight or branched alkylene;K is hydrogen or alkyl optionally substituted by substituent group A;P^(R0) is alkyl optionally substituted by substituent group A;P^(R1) is carbocyclyl group optionally substituted by substituent groupA, or heterocyclyl group optionally substituted by substituent group A;P^(R2) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A,heterocyclyl group optionally substituted by substituent group A,carbocyclylalkyl optionally substituted by substituent group A, orheterocyclylalkyl optionally substituted by substituent group A;P^(R3) is each independently hydrogen or alkyl;P^(R4) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A, orheterocyclyl group optionally substituted by substituent group A;P^(R6) is carbocyclyl group optionally substituted by substituent groupA, or heterocyclyl group optionally substituted by substituent group A;P^(R8) is alkyloxy optionally substituted by substituent group A;P^(R9) is alkyloxy optionally substituted by substituent group A,alkylamino optionally substituted by substituent group A, carbocyclyloxyoptionally substituted by substituent group A, heterocyclyloxyoptionally substituted by substituent group A, carbocyclyl aminooptionally substituted by substituent group A or heterocyclylaminooptionally substituted by substituent group A; andP^(R8) and P^(R9) may be taken together with an adjacent phosphorus atomto form heterocycle optionally substituted by substituent group A;Substituent group A; oxo, alkyl, alkylamino, carbocyclyl group,heterocyclyl group, alkylcarbonyl, halogen, hydroxy, alkylcarbonylamino,alkylcarbonyloxy, alkyloxycarbonyl, aryloxycarbonylalkyl,alkylaminocarbonyloxy, alkyloxy, nitro, azido, alkylsulfonyl andtrialkylsilyl.(13-1) The pharmaceutical composition according to (12), comprising thecompound or its pharmaceutically acceptable salt,wherein the group to form a prodrug is a group selected from thefollowing formula:—C(═O)—P^(R0),  a)—C(═O)—P^(R1),  b)—C(═O)—O—P^(R2),  g)—C(═O)—N(—K)(P^(R2)),  h)—C(═O)—O-L-O—P^(R2),  i)—C(P^(R3))₂—O—C(═O)—P^(R4),  l)—C(P^(R3))₂—O—C(═O)—O—P^(R4),  m)—C(P^(R3))₂—O—C(═O)—O-L-O—P^(R4),  o)—C(P^(R3))₂—C(P^(R3))₂—C(═O)—O—P^(R2),  x)—C(P^(R3))₂—N(—K)—C(═O)—O—P^(R2), and  y)—P(═O)(—P^(R8))(—P^(R9)),  z)wherein L is straight or branched alkylene;K is hydrogen, or alkyl optionally substituted by substituent group A;P^(R0) is alkyl optionally substituted by substituent group A;P^(R1) is carbocyclyl group optionally substituted by substituent groupA, or heterocyclyl group optionally substituted by substituent group A;P^(R2) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A,heterocyclyl group optionally substituted by substituent group A,carbocyclylalkyl optionally substituted by substituent group A, orheterocyclylalkyl optionally substituted by substituent group A;P^(R3) is each independently hydrogen or alkyl;P^(R4) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A, orheterocyclyl group optionally substituted by substituent group A;P^(R6) is carbocyclyl group optionally substituted by substituent groupA, or heterocyclyl group optionally substituted by substituent group A;P^(R8) is alkyloxy optionally substituted by substituent group A;P^(R9) is alkyloxy optionally substituted by substituent group A,alkylamino optionally substituted by substituent group A, carbocyclyloxyoptionally substituted by substituent group A, heterocyclyloxyoptionally substituted by substituent group A, carbocyclyl aminooptionally substituted by substituent group A or heterocyclylaminooptionally substituted by substituent group A; andP^(R8) and P^(R9) may be taken together with an adjacent phosphorus atomto form heterocycle optionally substituted by substituent group A;Substituent group A; oxo, alkyl, alkylamino, carbocyclyl, heterocyclyl,alkylcarbonyl, halogen, hydroxy, alkylcarbonylamino, alkylcarbonyloxy,alkyloxycarbonyl, aryloxycarbonylalkyl, alkylaminocarbonyloxy, alkyloxy,nitro, azido, alkylsulfonyl and trialkylsilyl.(14) The pharmaceutical composition according to (12), comprising thecompound or its pharmaceutically acceptable salt,wherein the group to form a prodrug is a following formula:—C(P^(R3))₂—O—C(═O)—O—P^(R4)  m)wherein P^(R3) is each independently hydrogen or alkyl; andP^(R4) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A, orheterocyclyl group optionally substituted by substituent group A;Substituent group A; oxo, alkyl, alkylamino, carbocyclyl group,heterocyclyl group, alkylcarbonyl, halogen, hydroxy, alkylcarbonylamino,alkylcarbonyloxy, alkyloxycarbonyl, aryloxycarbonylalkyl,alkylaminocarbonyloxy, alkyloxy, nitro, azido, alkylsulfonyl andtrialkylsilyl.(15) A pharmaceutical composition comprising a compound represented bythe

(16) A pharmaceutical composition comprising a compound represented bythe following formula or its pharmaceutically acceptable salt:

(17) A pharmaceutical composition comprising a compound represented bythe following formula or its pharmaceutically acceptable salt:

(18) A pharmaceutical composition comprising a compound represented bythe following formula or its pharmaceutically acceptable salt:

(19) A pharmaceutical composition comprising a compound represented bythe following formula or its pharmaceutically acceptable salt:

(20) A pharmaceutical composition comprising a compound represented bythe following formula or its pharmaceutically acceptable salt:

(21) A pharmaceutical composition comprising a compound represented bythe following formula or its pharmaceutically acceptable salt:

(22) The pharmaceutical composition according to any one of (1) to (21),which is an antiviral agent.(23) The pharmaceutical composition according to any one of (1) to (21),which is a cap-dependent endonuclease inhibitor.(24) The pharmaceutical composition according to any one of (1) to (21),which is used for shortening time to alleviation of influenza symptoms.(24-1) A method for shortening time to alleviation of influenzasymptoms, characterized in administering the compound of any one of (1)to (21) or its pharmaceutically acceptable salt.(24-2) A method for shortening time to alleviation of influenza symptomsto treat and/or prevent an influenza virus infectious disease,characterized in administering the compound of any one of (1) to (21) orits pharmaceutically acceptable salt.(24-3) The compound of any one of (1) to (21) or its pharmaceuticallyacceptable salt, for shortening time to alleviation of influenzasymptoms.(24-4) The compound of any one of (1) to (21) or its pharmaceuticallyacceptable salt, for shortening time to alleviation of influenzasymptoms to treat and/or prevent an influenza virus infectious disease.(25) The pharmaceutical composition according to any one of (1) to (21),which is used for reducing the influenza virus.(25-1) A method for reducing the influenza virus, characterized inadministering the compound of any one of (1) to (21) or itspharmaceutically acceptable salt.(25-2) A method for reducing the influenza virus to treat and/or preventan influenza virus infectious disease, characterized in administeringthe compound of any one of (1) to (21) or its pharmaceuticallyacceptable salt.(25-3) The compound of any one of (1) to (21) or its pharmaceuticallyacceptable salt, for reducing the influenza virus.(25-4) The compound of any one of (1) to (21) or its pharmaceuticallyacceptable salt, for reducing the influenza virus to treat and/orprevent an influenza virus infectious disease.(26) A crystal of a compound of the following formula:

(27) The crystal according to (26), having two or more peaks indiffraction angles (2θ) selected from 8.6±0.2°, 14.1±0.2°, 17.4±0.2°,20.0±0.2°, 24.0±0.2°, 26.3±0.2°, 29.6±0.2°, and 35.4±0.2° in an X-raypowder diffraction spectrum.(28) The crystal according to (26), having peaks in diffraction angles(2θ) of: 8.6±0.2°, 14.1±0.2°, 17.4±0.2°, 20.0±0.2°, 24.0±0.2°,26.3±0.2°, 29.6±0.2° and 35.4±0.2° in an X-ray powder diffractionspectrum.(29) The crystal according to (26), wherein an X-ray powder diffractionspectrum of the crystal is substantially identical with FIG. 3.(30) A pharmaceutical composition comprising the crystal of any one of(26) to (29).(31) The pharmaceutical composition according to (30), which is anantiviral agent.(32) The pharmaceutical composition according to (30), which is acap-dependent endonuclease inhibitor.(33) A pharmaceutical composition comprising the crystal of any one of(26) to (29), which is used for shortening time to alleviation ofinfluenza symptoms.(33-1) A method for shortening time to alleviation of influenzasymptoms, characterized in administering the crystal of any one of (26)to (29).(33-2) A method for shortening time to alleviation of influenza symptomsto treat and/or prevent an influenza virus infectious disease,characterized in administering the crystal of any one of (26) to (29).(33-3) The crystal according to any one of (26) to (29), for shorteningtime to alleviation of influenza symptoms.(33-4) The crystal according to any one of (26) to (29), for shorteningtime to alleviation of influenza symptoms to treat and/or prevent aninfluenza virus infectious disease.(34) A pharmaceutical composition comprising the crystal of any one of(26) to (29), which is used for reducing the influenza virus.(34-1) A method for reducing the influenza virus, characterized inadministering the crystal of any one of (26) to (29).(34-2) A method for reducing the influenza virus to treat and/or preventan influenza virus infectious disease, characterized in administeringthe crystal of any one of (26) to (29).(34-3) The crystal according to any one of (26) to (29), for reducingthe influenza virus.(35) The pharmaceutical composition according to any one of (1) to (21)and (30) to (34), for oral administration.(36) The pharmaceutical composition according to (35), which is atablet, powder, granule, capsule, pill, film, suspension, emulsion,elixir, syrup, lemonade, spirit, aromatic water, extract, decoction ortincture.(37) The pharmaceutical composition according to (35), which is asugar-coated tablet, film-coated tablet, enteric-coated tablet,sustained-release tablet, troche tablet, sublingual tablet, buccaltablet, chewable tablet, orally disintegrated tablet, dry syrup, softcapsule, micro capsule or sustained-release capsule.(38) The pharmaceutical composition according to any one of (1) to (21)and (30) to (34), for parenteral administration.(39) The pharmaceutical composition according to (38), for dermal,subcutaneous, intravenous, intraarterial, intramuscular,intraperitoneal, transmucosal, inhalation, transnasal, ophthalmic, innerear or vaginal administration.(40) The pharmaceutical composition according to (38) or (39), which isinjection, infusion, eye drop, nose drop, ear drop, aerosol, inhalation,lotion, impregnation, liniment, mouthwash, enema, ointment, plaster,jelly, cream, patch, cataplasm, external powder or suppository.(41) The pharmaceutical composition according to any one of (1) to (21)and (30) to (34), for a pediatric or geriatric patient.(42) A pharmaceutical composition consisting of a combination of thepharmaceutical composition according to any one of (1) to (21) and (30)to (34) and Neuraminidase inhibitor, RNA-dependent RNA polymeraseinhibitor, M2 protein inhibitor, PB2 Cap binding inhibitor, an anti-HAantibody or immunological agent.(43) A pharmaceutical composition comprising the pharmaceuticalcomposition according to any one of (1) to (21) and (30) to (34), for acombination therapy with Neuraminidase inhibitor, RNA-dependent RNApolymerase inhibitor, M2 protein inhibitor, PB2 Cap binding inhibitor,an anti-HA antibody or immunological agent.

Note that (13) above encompasses (13-1) above.

The present invention provides a method for treating or preventing aninfluenza virus infectious disease using the compounds (parent compoundsand/or prodrug compounds) used in the present invention, andpharmaceutical compositions used therein. The parent compounds areeffective as anti-influenza agents or intermediates of the prodrugcompounds.

Effect of the Invention

The compounds (parent compounds and/or prodrugs) used in the presentinvention have inhibitory activity on cap-dependent endonuclease. Morepreferred compound is a prodrug, and the prodrug becomes a parentcompound having an inhibitory activity on cap-dependent endonuclease invivo after administration, thus is effective as a therapeutic agentand/or preventive agent for influenza virus infectious disease.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a result of measuring the plasma concentration of CompoundIII-2, after oral administration of prodrug Compound II-6, the parentcompound of which is Compound III-2, to rat under non-fastingconditions.

FIG. 2 is a result of measuring the plasma concentration of CompoundII-6, after oral administration of prodrug Compound II-6, the parentcompound of which is Compound III-2, to rat under non-fastingconditions.

FIG. 3 is powder X-ray diffraction data of I-form crystals of CompoundII-6. The horizontal axis indicates 20, and the vertical axis indicatesintensity.

FIG. 4 is powder X-ray diffraction data of II-form crystals of CompoundII-6.

FIG. 5 is powder X-ray diffraction data of III-form crystals of CompoundII-6.

BEST MODE FOR CARRYING OUT THE INVENTION

The meaning of each term used in the present description is explainedbelow. Each term is used in a unified sense, and is used in the samesense when used alone, or when used in combination of other term.

The term of “consisting of” means having only components.

The term of “comprising” means not restricting with components and notexcluding undescribed factors.

“Optionally substituted by substituent group A” means that an arbitraryposition may be substituted by one, two or more same or differentsubstituents selected from substituent group A.

“Prodrug” in the present description refers to a compound represented byformula (II) in the following reaction formula:

wherein each symbol is same as the above,or its pharmaceutically acceptable salt, and means a compound showingcap-dependant endonuclease (CEN) inhibitory activity and/or CPEinhibitory effect by being converted into a compound represented byformula (III) by a decomposition reaction caused by drug-metabolizingenzymes, hydrolases, gastric acids, enterobacteria, etc. underphysiological conditions in vivo.

The prodrug more preferably means a compound in which bioavailabilityand/or AUC (area under the blood concentration curve) in in vivoadministration is improved more than those of the compound representedby formula (III).

Therefore, the prodrug is efficiently absorbed into the body in thestomach and/or intestines after in vivo administration (for example,oral administration), then converted into the compound represented byformula (III). Thus, the prodrug preferably shows an effect of treatingand/or preventing influenza higher than the compound represented byformula (III).

One embodiment of the “group represented by

“wherein each definition has the same meaning as described (1),is a group represented by formula:

wherein R², R³, R⁴ and R⁵ are each independently hydrogen or fluorine;the number of fluorine atoms of R², R³, R⁴ and R⁵ is 1 or 2.

Another embodiment is a group represented by formula:

and a group represented by formula:

is preferable, and a group represented by formula:

is especially preferable.

“Group to form a prodrug” in the present description refers to a “P^(R)”group in the formula (II), in the following reaction formula:

wherein each symbol is same as the above,and —OP^(R) group is converted into —OH group in the formula (III) by adecomposition reaction caused by drug-metabolizing enzymes, hydrolases,gastric acids, enterobacteria, etc. under physiological conditions invivo.

The “group to form a prodrug” more preferably means a group thatimproves bioavailability and/or AUC (area under the blood concentrationcurve) of the compound represented by formula (III) by being added tothe compound represented by formula (III).

Examples of the group to form a prodrug include the groups described inProg. Med. 5: 2157-2161 (1985) and Supplied by The British Library—“Theworld's Knowledge”.

The “group to form a prodrug” in —OP^(R) group in the formula (I) or(II) may be a group converted into —OH group in vivo, and examplespreferably include a group selected from the following formula a) toac).—C(═O)—P^(R0),  a)—C(═O)—P^(R1),  b)—C(═O)-L-P^(R1),  c)—C(═O)-L-O—P^(R1),  d)—C(═O)-L-O-L-O—P^(R1),  e)—C(═O)-L-O—C(═O)—P^(R1),  f)—C(═O)—O—P^(R2),  g)—C(═O)—N(—K)(P^(R2)),  h)—C(═O)—O-L-O—P^(R2),  i)—C(P^(R3))₂—O—P^(R4),  j)—C(P^(R3))₂—O-L-O—P^(R4),  k)—C(P^(R3))₂—O—C(═O)—P^(R4),  l)—C(P^(R3))₂—O—C(═O)—O—P^(R4),  m)—C(P^(R3))₂—O—C(═O)—N(—K)—P^(R4),  n)—C(P^(R3))₂O—C(═O)—O-L-O—P^(R4),  o)—C(P^(R3))₂—O—C(═O)—O-L-N(P^(R4))₂,  p)—C(P^(R3))₂—O—C(═O)—N(—K)-L-O—P^(R4),  q)—C(P^(R3))₂—O—C(═O)—N(—K)-L-N(P^(R4))₂,  r)—C(P^(R3))₂—O—C(═O)—O-L-O-L-O—P^(R4),  s)—C(P^(R3))₂—O—C(═O)—O-L-N(—K)—C(═O)—P^(R4),  t)—C(P^(R3))₂O—P(═O)(P^(R5))₂,  u)—C(P^(R3))₂—P^(R6),  v)—C(═N⁺(P^(R7))₂)(—N(P^(R7))₂),  w)—C(P^(R3))₂—C(P^(R3))₂—C(═O)—O—P^(R2),  x)—C(P^(R3))₂—N(—K)—C(═O)—O—P^(R2),  y)—P(═O)(—P^(R8))(—P^(R9)),  z)—S(═O)₂—P^(R10),  aa)—P^(R11), and  ab)—C(P^(R3))₂—C(P^(R3))₂—O—P^(R2),  ac)wherein L is straight or branched alkylene, or straight or branchedalkenylene;K is hydrogen, or alkyl optionally substituted by substituent group A;P^(R0) is alkyl optionally substituted by substituent group A, oralkenyl optionally substituted by substituent group A;P^(R1) is carbocyclyl group optionally substituted by substituent groupA, heterocyclyl group optionally substituted by substituent group A,alkylamino optionally substituted by substituent group A, oralkylsulfanyl optionally substituted by substituent group A;P^(R2) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A,heterocyclyl group optionally substituted by substituent group A,carbocyclylalkyl optionally substituted by substituent group A,heterocyclylalkyl optionally substituted by substituent group A ortrialkylsilyl;P^(R3) is each independently hydrogen or alkyl;P^(R4) is each independently alkyl optionally substituted by substituentgroup A, carbocyclyl group optionally substituted by substituent groupA, heterocyclyl group optionally substituted by substituent group A,alkylamino optionally substituted by substituent group A,carbocyclylalkyl optionally substituted by substituent group A,heterocyclylalkyl optionally substituted by substituent group A, ortrialkylsilyl;P^(R5) is each independently hydroxy or OBn;P^(R6) is carbocyclyl group optionally substituted by substituent groupA, or heterocyclyl group optionally substituted by substituent group A;P^(R7) is each independently alkyl optionally substituted by substituentgroup A;P^(R8) is alkyloxy optionally substituted by substituent group A;P^(R9) is alkyloxy optionally substituted by substituent group A,alkylamino optionally substituted by substituent group A, carbocyclyloxyoptionally substituted by substituent group A, heterocyclyloxyoptionally substituted by substituent group A, carbocyclyl aminooptionally substituted by substituent group A or heterocyclylaminooptionally substituted by substituent group A;P^(R8) and P^(R9) may be taken together with an adjacent phosphorus atomto form heterocycle optionally substituted by substituent group A;P^(R10) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A,heterocyclyl group optionally substituted by substituent group A,carbocyclylalkyl optionally substituted by substituent group A orheterocyclylalkyl optionally substituted by substituent group A; andP^(R11) is alkyl optionally substituted by substituent group A, alkenyloptionally substituted by substituent group A, carbocyclyl groupoptionally substituted by substituent group A, or heterocyclyl groupoptionally substituted by substituent group A;Substituent group A; oxo, alkyl, hydroxyalkyl, amino, alkylamino,carbocyclyl, heterocyclyl, carbocyclylalkyl, alkylcarbonyl, halogen,hydroxy, carboxy, alkylcarbonylamino, alkylcarbonylaminoalkyl,alkylcarbonyloxy, alkyloxycarbonyl, alkyloxycarbonylalkyl,alkyloxycarbonyloxy, alkylaminocarbonyloxy, alkylaminoalkyl, alkyloxy,cyano, nitro, azido, alkylsulfonyl, trialkylsilyl and phospho.

“Converted into a prodrug” in the present description means that, asshown in the following reaction formula:

wherein each symbol is same as the above,a hydroxy group in the formula (III) or its pharmaceutically acceptablesalt is converted into —OP^(R) group.

“Parent compound” in the present description means a compound to be asource before synthesizing the “prodrug” and/or a compound released fromthe “prodrug” by the reaction by enzymes, a gastric acid, and the likeunder physiological conditions in vivo, and specifically means acompound shown by the formula (III), or pharmaceutically acceptable saltthereof or a solvate thereof.

The term “halogen” includes a fluorine atom, a chlorine atom, a bromineatom and an iodine atom. A fluorine atom and a chlorine atom areespecially preferable.

The term “alkyl” includes a C1 to C15, preferably C1 to C10, morepreferably C1 to C6 and further preferably C1 to C4 linear or branchedhydrocarbon group. Examples include methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, secbutyl, tert-butyl, n-pentyl, isopentyl, neopentyl,n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl,n-decyl and the like.

A preferred embodiment of “alkyl” is methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, secbutyl, tert-butyl or n-pentyl. A more preferredembodiment is methyl, ethyl, n-propyl, isopropyl or tert-butyl.

The term “alkenyl” includes a C2 to C15, preferably a C2 to C10, morepreferably a C2 to C6 and further preferably a C2 to C4 linear orbranched hydrocarbon group having one or more double bond(s) at anyposition(s). Examples include vinyl, allyl, propenyl, isopropenyl,butenyl, isobutenyl, prenyl, butadienyl, pentenyl, isopentenyl,pentadienyl, hexenyl, isohexenyl, hexadienyl, heptenyl, octenyl,nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl,pentadecenyl and the like.

A preferred embodiment of “alkenyl” is vinyl, allyl, propenyl,isopropenyl or butenyl.

The term “alkylene” includes a C1 to C15, preferably a C1 to C10, morepreferably a C1 to C6 and further preferably a C1 to C4 liner orbranched bivalent hydrocarbon group. Examples include methylene,ethylene, trimethylene, propylene, tetramethylene, pentamethylene,hexamethylene and the like.

The term “alkenylene” includes a C2 to C15, preferably a C2 to C10, morepreferably a C2 to C6 and further preferably a C2 to C4 liner orbranched bivalent hydrocarbon group having one or more double bond(s) atany position(s). Examples include vinylene, prenylene, butenylene,pentenylene and the like.

The term “hydroxyalkyl” means a group wherein one or more hydroxylgroup(s) is replaced with hydrogen atom(s) attached to a carbon atom(s)of the above “alkyl”. Examples include hydroxymethyl, 1-hydroxyethyl,2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 1,2-hydroxyethyl andthe like.

A preferred embodiment of “hydroxyalkyl” is hydroxymethyl.

The term “alkyloxy” means a group wherein the above “alkyl” is bonded toan oxygen atom. Examples include methyloxy, ethyloxy, n-propyloxy,isopropyloxy, n-butyloxy, tert-butyloxy, isobutyloxy, sec-butyloxy,pentyloxy, isopentyloxy, hexyloxy and the like.

A preferred embodiment of “alkyloxy” is methyloxy, ethyloxy,n-propyloxy, isopropyloxy or tert-butyloxy.

The term “haloalkyl” means a group wherein one or more “halogen”described above is bonded to the above “alkyl”. Examples includemonofluoromethyl, monofluoroethyl, monofluoropropyl,2,2,3,3,3-pentafluoropropyl, monochloromethyl, trifluoromethyl,trichloromethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloroethyl,1,2-dibromoethyl, 1,1,1-trifluoropropan-2-yl and the like.

A preferred embodiment of “haloalkyl” is trifluoromethyl ortrichloromethyl.

The term “alkylcarbonyl” means a group wherein the above “alkyl” isbonded to a carbonyl group. Examples include methylcarbonyl,ethylcarbonyl, propylcarbonyl, isopropylcarbonyl, tert-butylcarbonyl,isobutylcarbonyl, sec-butylcarbonyl, pentylcarbonyl, isopenthylcarbonyl,hexylcarbonyl and the like.

A preferred embodiment of “alkylcarbonyl” is methylcarbonyl,ethylcarbonyl or n-prop ylcarbonyl.

The term “alkylamino” means a group wherein one or two hydrogen atom(s)attached to a nitrogen atom of an amino group is replaced with the above“alkyl”. Two alkyl groups may be the same or different. Examples includemethylamino, ethylamino, isopropylamino, dimethylamino, diethylamino,N,N-diisopropylamino, N-methyl-N-ethylamino, N-isopropyl-N-ethylaminoand the like.

A preferred embodiment of “alkylamino” is methylamino, ethylamino,dimethylamino or diethylamino.

The term “alkylaminoalkyl” means a group wherein the above “alkylamino”is bonded to the above “alkyl”.

The term “alkylaminocarbonyl” means a group wherein the above“alkylamino” is bonded to a carbonyl group.

The term “alkylaminocarbonyloxy” means a group wherein the above“alkylaminocarbonyl” is bonded to an oxygen atom.

The term “alkylcarbonylamino” means a group wherein the above“alkylcarbonyl” is replaced with a hydrogen atom bonded to a nitrogenatom of an amino group. Examples include methylcarbonylamino,ethylcarbonylamino, propylcarbonylamino, isopropylcarbonylamino,tert-butylcarbonylamino, isobutylcarbonylamino, sec-butylcarbonylaminoand the like.

A preferred embodiment of “alkylcarbonylamino” is methylcarbonylamino orethylcarbonylamino.

The term “alkylcarbonyloxy” means a group wherein the above“alkylcarbonyl” is bonded to an oxygen atom. Examples includemethylcarbonyloxy, ethylcarbonyloxy, propylcarbonyloxy,isopropylcarbonyloxy, tert-butylcarbonyloxy, isobutylcarbonyloxy,secbutyl carbonyloxy and the like.

A preferred embodiment of “alkylcarbonyloxy” is methylcarbonyloxy orethylcarbonyloxy.

The term “alkylcarbonylaminoalkyl” means a group wherein the above“alkylcarbonylamino” is bonded to the above “alkyl”.

The term “alkyloxycarbonyl” means a group wherein the above “alkyloxy”is bonded to a carbonyl group. Examples include methyloxycarbonyl,ethyloxycarbonyl, propyloxycarbonyl, isopropyloxycarbonyl,tert-butyloxycarbonyl, isobutyloxycarbonyl, sec-butyloxycarbonyl,penthyloxycarbonyl, isopenthyloxycarbonyl, hexyloxycarbonyl and thelike.

A preferred embodiment of “alkyloxycarbonyl” is methyloxycarbonyl,ethyloxycarbonyl or propyloxycarbonyl.

The term “aryloxycarbonylalkyl” means a group wherein the above“alkyloxycarbonyl” is bonded to the above “alkyl”.

The term “alkyloxycarbonyloxy” means a group wherein the above“alkyloxycarbonyl” is bonded to an oxygen atom.

The term “alkylsulfanyl” means a group wherein the above “alkyl” isreplaced with a hydrogen atom bonded to a sulfur atom of a sulfanylgroup. Examples include methylsulfanyl, ethylsulfanyl, n-propylsulfanyl,isopropylsulfanyl and the like.

The term “alkylsulfonyl” means a group wherein the above “alkyl” isbonded to a sulfonyl group. Examples include methylsulfonyl,ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, tert-butylsulfonyl,isobutylsulfonyl, sec-butylsulfonyl and the like.

A preferred embodiment of “alkylsulfonyl” is methylsulfonyl orethylsulfonyl.

The term “trialkylsilyl” means a group wherein three of the above“alkyl” are bonded to a silicon atom. Three alkyl groups may be the sameor different. Examples include trimethylsilyl, triethylsilyl,tert-butyldimethylsilyl and the like.

The term “carbocyclyl group” means C3 to C20 preferably C3 to C16, morepreferably C4 to C12 cyclic hydrocarbon group and includes aromaticcarbocyclyl and non-aromatic carbocyclyl.

The term “aromatic carbocyclyl” means a cyclic aromatic hydrocarbongroup which is monocyclic or polycyclic having two or more rings.Examples include phenyl, naphthyl, anthryl, phenanthryl and the like.

A preferred embodiment of “aromatic carbocyclyl” is phenyl, 1-naphthylor 2-naphthyl. Another embodiment of “aromatic carbocyclyl” is phenyl,

The term “non-aromatic carbocyclyl” means a cyclic saturated hydrocarbongroup or a cyclic unsaturated non-aromatic hydrocarbon group, which ismonocyclic or polycyclic having two or more rings. Examples of the“non-aromatic carbocyclyl”, which is polycyclic having two or morerings, include a fused ring group wherein a non-aromatic carbocyclyl,which is monocyclic or polycyclic having two or more rings, is fusedwith a ring of the above “aromatic carbocyclyl”.

In addition, examples of the “non-aromatic carbocyclyl” also include agroup having a bridge or a group to form a spiro ring as follows:

The non-aromatic carbocyclyl which is monocyclic is preferably C3 toC16, more preferably C3 to C12 and further preferably C3 to C8carbocyclyl. Examples include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclohexadienyl and the like.

Examples of non-aromatic carbocyclyl, which is polycyclic having two ormore rings, include indanyl, indenyl, acenaphthyl, tetrahydronaphthyl,fluorenyl and the like.

The term “carbocycle” means C3 to C20 preferably C3 to C16, morepreferably C4 to C12 cyclic hydrocarbon and includes aromatic carbocycleand non-aromatic carbocycle.

The term “aromatic carbocycle” means a cyclic aromatic hydrocarbon whichis monocyclic or polycyclic having two or more rings. Examples includebenzene ring, naphthalene ring, anthracene ring, phenanthrene ring andthe like.

A preferred embodiment of “aromatic carbocycle” is benzene ring andnaphthalene ring are exemplified. Another embodiment of “aromaticcarbocycle” is benzene ring.

The term of “non-aromatic carbocycle” means a saturated carbocycle or anunsaturated non-aromatic carbocycle which is monocyclic or polycyclichaving two or more rings. Examples of the “non-aromatic carbocycle”which is polycyclic having two or more rings, include a fused ringwherein a non-aromatic carbocycle, which is monocyclic or polycyclichaving two or more rings, is fused with a ring of the above “aromaticcarbocycle”.

In addition, examples of the “non-aromatic carbocycle” also include acycle having a bridge or a cycle to form a spiro ring as follows:

The non-aromatic carbocycle which is monocyclic is preferably C3 to C16,more preferably C3 to C12 and further preferably C3 to C8 carbocycle.Examples include cyclopropane, cyclobutane, cyclopentane, cyclohexane,cycloheptane, cyclooctane, cyclononane, cyclodecane, cyclopropene,cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclohexadiene andthe like.

Examples of non-aromatic carbocycle, which is polycyclic having two ormore rings, include indane, indene, acenaphthalene,tetrahydronaphthalene, fluorine and the like are exemplified.

The term “heterocyclyl group” includes an aromatic cyclyl and anon-aromatic heterocyclyl, which is containing one or more ofheteroatom(s) selected independently from O, S and N.

The term “aromatic heterocyclyl” means an aromatic cyclyl, which ismonocyclic or polycyclic having two or more rings, containing one ormore of heteroatom(s) selected independently from O, S and N.

Examples of “aromatic heterocyclyl”, which is polycyclic having two ormore rings, include a fused ring group wherein an aromatic heterocyclyl,which is monocyclic or polycyclic having two or more rings, is fusedwith a ring of the above “aromatic carbocyclyl”.

The aromatic heterocyclyl, which is monocyclic, is preferably a 5- to8-membered and more preferably 5- to 6-membered ring. Examples includepyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazolyl, triazinyl, tetrazolyl, furyl, thienyl, isoxazolyl,oxazolyl, oxadiazolyl, isothiazolyl, thiazolyl, thiadiazolyl and thelike.

Examples of aromatic heterocyclyl, which is bicyclic, include indolyl,isoindolyl, indazolyl, indolizinyl, quinolinyl, isoquinolinyl,cinnolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, quinoxalinyl,purinyl, pteridinyl, benzimidazolyl, benzisoxazolyl, benzoxazolyl,benzoxadiazolyl, benzisothiazolyl, benzothiazolyl, benzothiadiazolyl,benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, imidazopyridyl,triazolopyridyl, imidazothiazolyl, pyrazinopyridazinyl, oxazolopyridyl,thiazolopyridyl and the like.

Examples of aromatic heterocyclyl, which is polycyclic having three ormore rings, include carbazolyl, acridinyl, xanthenyl, phenothiazinyl,phenoxathiinyl, phenoxazinyl, dibenzofuryl and the like.

The term “non-aromatic heterocyclyl” means a non-aromatic cyclyl, whichis monocyclic or polycyclic having two or more rings, containing one ormore heteroatom(s) selected independently from O, S and N.

Examples of “non-aromatic heterocyclyl”, which is polycyclic having twoor more rings, include a fused ring group wherein a non-aromaticheterocycle, which is monocyclic or polycyclic having two or morering(s), is fused with a ring of the above “aromatic carbocyclyl”,“non-aromatic carbocyclyl” and/or “aromatic heterocyclyl”.

In addition, examples of the “non-aromatic heterocyclyl” also include agroup having a bridge or a group to form a spiro ring as follows:

The non-aromatic heterocyclyl, which is monocyclic, is preferably a 3-to 8-membered and more preferably 5- to 6-membered ring. Examplesinclude dioxanyl, thiiranyl, oxiranyl, oxetanyl, oxathiolanyl,azetidinyl, thianyl, thiazolidinyl, pyrrolidinyl, pyrrolinyl,imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidinyl,piperazinyl, morpholinyl, morpholino, thiomorpholinyl, thiomorpholino,dihydropyridinyl, tetrahydropyridinyl, tetrahydrofuryl,tetrahydropyranyl, dihydrothiazolinyl, tetrahydrothiazolinyl,tetrahydroisothiazolinyl, dihydrooxazinyl, hexahydroazepinyl, tetrahydrodiazepinyl, tetrahydropyridazinyl, hexahydropyrimidinyl, dioxolanyl,dioxazinyl, aziridinyl, dioxolinyl, oxepanyl, thiolanyl, thiinyl,thiazinyl and the like.

Examples of non-aromatic heterocyclyl, which is polycyclic having two ormore rings, include indolinyl, isoindolinyl, chromanyl, isochromanyl andthe like.

The term “heterocycle” includes an aromatic cycle and a non-aromaticheterocycle, which is containing one or more of heteroatom(s) selectedindependently from O, S and N.

The term of “aromatic heterocycle” means an aromatic cycle which ismonocyclic or polycyclic having two or more rings, containing one ormore of heteroatom(s) selected independently from O, S and N.

Examples of “aromatic heterocycle”, which is polycyclic having two ormore rings, include a fused ring wherein an aromatic heterocycle, whichis monocyclic or polycyclic having two or more rings, is fused with aring of the above “aromatic carbocycle”.

The aromatic heterocycle, which is monocyclic, is preferably a 5- to8-membered and more preferably 5- to 6-membered ring. Examples includepyrrole, imidazole, pyrazole, pyridine, pyridazine, pyrimidine,pyrazine, triazole, triazine, tetrazole, furan, thiophene, isoxazole,oxazole, oxadiazole, isothiazole, thiazole, thiadiazole and the like.

Examples of aromatic heterocycle, which is bicyclic, include indoline,isoindoline, indazorin, indolizine, quinoline, isoquinoline, cinnoline,phthalazine, quinazoline, naphthyridine, quinoxaline, purine, pteridine,benzimidazole, benzisoxazole, benzoxazole, benzoxadiazole,benzisothiazole, benzothiazole, benzothiadiazole, benzofuran,isobenzofuran, benzothiophene, benzotriazole, imidazopyridine,triazolopyridine, imidazothiazole, pyrazinopyridazine, oxazolopyridine,thiazolopyridine and the like.

Examples of aromatic heterocycle, which is polycyclic having three ormore rings, include carbazole, acridine, xanthene, phenothiazine,phenoxathiin, phenoxazine, dibenzofuran and the like.

The term “non-aromatic heterocycle” means a non-aromatic cycle, which ismonocyclic or polycyclic having two or more rings, containing one ormore of heteroatom(s) selected independently from O, S and N.

Examples of “non-aromatic heterocycle”, which is polycyclic having twoor more rings, include a fused ling wherein a non-aromatic heterocycle,which is monocyclic or polycyclic having two or more ring(s), is fusedwith a ring of the above “aromatic carbocycle”, “non-aromaticcarbocycle” and/or “aromatic heterocycle”.

In addition, examples of “non-aromatic heterocycle” also include a cyclehaving a bridge or a cyclo to form a spiro ring as follows:

The non-aromatic heterocycle, which is monocyclic, is preferably a 3- to8-membered and more preferably 5- to 6-membered ring. Examples includedioxane, thiirane, oxirane, oxetane, oxathiolane, azetidine, thiane,thiazolidine, pyrrolidine, pyrroline, imidazolidine, imidazoline,pyrazolidine, pyrazoline, piperidine, piperazine, morpholine,thiomorpholine, dihydropyridine, tetrahydropyridine, tetrahydrofuran,tetrahydropyran, dihydrothiazoline, tetrahydrothiazoline,tetrahydroisothiazoline, dihydrooxazine, hexahydroazepine,tetrahydrodiazepine, tetrahydropyridazine, hexahydropyrimidine,dioxolane, dioxazine, aziridine, dioxoline, oxepane, thiolane, thiazineand the like.

Examples of non-aromatic heterocycle, which is polycyclic having two ormore rings, include indoline, isoindoline, chroman, isochroman and thelike.

The “carbocycle” part of “carbocyclylalkyl”, “carbocyclyloxy” or“carbocyclylamino” is same as the above “carbocycle”.

The “heterocycle” part of “heterocyclylalkyl”, “heterocyclyloxy” or“heterocyclylamino” is same as the above “heterocycle”.

The compounds used in the present invention are characterized in thatthe compounds isolated by optical resolution of tricyclic compoundssubstituted by the other tricyclic group improve cap-dependentendonuclease inhibitory activity.

The compounds used in the present invention are also characterized inthat the compounds are efficiently absorbed into the body afteradministration (for example, oral administration), and showing highefficacy by introducing a group to form a prodrug.

One or more hydrogen, carbon and/or other atoms in the compounds used inthe present invention may be replaced with isotopes of hydrogen, carbonand/or other atoms respectively. Examples of isotopes include hydrogen,carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine andchlorine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S,¹⁸F, ¹²³I and ³⁶Cl respectively. The compounds used in the presentinvention include compounds replaced with these isotopes. The compoundsreplaced with the above isotopes are useful as medicines and include allof radiolabeled compounds used in the present invention. A “method ofradiolabeling” in the manufacture of the “radiolabeled compounds” isencompassed by the present invention, and the “radiolabeled compounds”are useful for studies on metabolized drug pharmacokinetics, studies onbinding assay and/or diagnostic tools.

A radiolabeled compound used in the present invention can be preparedusing well-known methods in this field of the invention. For example, atritium-labeled compound used in the present invention can be preparedby introducing a tritium to a certain compound used in the presentinvention, through a catalytic dehalogenation reaction using a tritium.This method comprises reacting with an appropriately-halogenatedprecursor of the compound used in the present invention with tritium gasin the presence of an appropriate catalyst, such as Pd/C, and in thepresence or absent of a base. The other appropriate method of preparinga tritium-labeled compound can be referred to “Isotopes in the Physicaland Biomedical Sciences, Vol. 1, Labeled Compounds (Part A), Chapter 6(1987)”. A ¹⁴C-labeled compound can be prepared by using a raw materialhaving ¹⁴C.

The pharmaceutically acceptable salts of the compounds used in thepresent invention include, for example, salts with alkaline metal (e.g.,lithium, sodium, potassium or the like), alkaline earth metal (e.g.,calcium, barium or the like), magnesium, transition metal (e.g., zinc,iron or the like), ammonia, organic bases (e.g., trimethylamine,triethylamine, dicyclohexylamine, ethanolamine, diethanolamine,triethanolamine, meglumine, ethylenediamine, pyridine, picoline,quinoline or the like) or amino acids, or salts with inorganic acids(e.g., hydrochloric acid, sulfuric acid, nitric acid, carbonic acid,hydrobromic acid, phosphoric acid, hydroiodic acid or the like) ororganic acids (e.g., formic acid, acetic acid, propionic acid,trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalicacid, maleic acid, fumaric acid, mandelic acid, glutaric acid, malicacid, benzoic acid, phthalic acid, ascorbic acid, benzenesulfonic acid,p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid or thelike). Especially, salts with hydrochloric acid, sulfuric acid,phosphoric acid, tartaric acid, methanesulfonic acid and the like areincluded. These salts can be formed by the usual methods.

The compounds used in the present invention or its pharmaceuticallyacceptable salts may form solvates (e.g., hydrates or the like) and/orcrystal polymorphs. The present invention encompasses those varioussolvates and crystal polymorphs. “Solvates” may be those wherein anynumbers of solvent molecules (e.g., water molecules or the like) arecoordinated with the compounds used in the present invention. When thecompounds used in the present invention or its pharmaceuticallyacceptable salts are allowed to stand in the atmosphere, the compoundsmay absorb water, resulting in attachment of adsorbed water or formationof hydrates. Recrystallization of the compounds used in the presentinvention or its pharmaceutically acceptable salts may produce crystalpolymorphs.

The group to form a prodrug is preferably a group converted into OHgroup by action of drug-metabolizing enzymes, hydrolases, gastric acids,and/or enterobacteria, after in vivo administration (for example, oraladministration).

Examples of more preferred embodiment of the group to form a prodruginclude a group selected from the following formulae a) to ac).—C(═O)—P^(R0),  a)—C(═O)—P^(R1),  b)—C(═O)-L-P^(R1),  c)—C(═O)-L-O—P^(R1),  d)—C(═O)-L-O-L-O—P^(R1),  e)—C(═O)-L-O—C(═O)—P^(R1),  f)—C(═O)—O—P^(R2),  g)—C(═O)—N(—K)(P^(R2)),  h)—C(═O)—O-L-O—P^(R2),  i)—C(P^(R3))₂—O—P^(R4),  j)—C(P^(R3))₂—O-L-O—P^(R4),  k)—C(P^(R3))₂—O—C(═O)—P^(R4),  l)—C(P^(R3))₂—O—C(═O)—O—P^(R4),  m)—C(P^(R3))₂—O—C(═O)—N(—K)—P^(R4),  n)—C(P^(R3))₂—O—C(═O)—O-L-O—P^(R4),  o)—C(P^(R3))₂—O—C(═O)—O-L-N(P^(R4))₂,  p)—C(P^(R3))₂—O—C(═O)—N(—K)-L-O—P^(R4),  q)—C(P^(R3))₂—O—C(═O)—N(—K)-L-N(P^(R4))₂,  r)—C(P^(R3))₂—O—C(═O)—O-L-O-L-O—P^(R4),  s)—C(P^(R3))₂—O—C(═O)—O-L-N(—K)—C(═O)—P^(R4),  t)—C(P^(R3))₂—O—P(═O)(—P^(R5))₂,  u)—C(P^(R3))₂—P^(R6),  v)—C(═N⁺(P^(R7))₂)(—N(P^(R7))₂),  w)—C(P^(R3))₂—C(P^(R3))₂—C(═O)—O—P^(R2),  x)—C(P^(R3))₂—N(—K)—C(═O)—O—P^(R2),  y)—P(═O)(—P^(R8))(—P^(R9)),  z)—S(═O)₂—P^(R10),  aa)—P^(R11), and  ab)—C(P^(R3))₂—C(P^(R3))₂—O—P^(R2),  ac)wherein L is straight or branched alkylene, or straight or branchedalkenylene;K is hydrogen, or alkyl optionally substituted by substituent group A;P^(R0) is alkyl optionally substituted by substituent group A, oralkenyl optionally substituted by substituent group A;P^(R1) is carbocyclyl group optionally substituted by substituent groupA, heterocyclyl group optionally substituted by substituent group A,alkylamino optionally substituted by substituent group A, oralkylsulfanyl optionally substituted by substituent group A;P^(R2) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A,heterocyclyl group optionally substituted by substituent group A,carbocyclylalkyl optionally substituted by substituent group A,heterocyclylalkyl optionally substituted by substituent group A ortrialkylsilyl optionally substituted by substituent group A;P^(R3) is each independently hydrogen or alkyl;P^(R4) is each independently alkyl optionally substituted by substituentgroup A, carbocyclyl group optionally substituted by substituent groupA, heterocyclyl group optionally substituted by substituent group A,alkyl amino optionally substituted by substituent group A,carbocyclylalkyl optionally substituted by substituent group A,heterocyclylalkyl optionally substituted by substituent group A, ortrialkylsilyl;P^(R5) is each independently hydroxy or OBn;P^(R6) is carbocyclyl group optionally substituted by substituent groupA, or heterocyclyl group optionally substituted by substituent group A;P^(R7) is each independently alkyl optionally substituted by substituentgroup A;P^(R8) is alkyloxy optionally substituted by substituent group A;P^(R9) is alkyloxy optionally substituted by substituent group A,alkylamino optionally substituted by substituent group A, carbocyclyloxyoptionally substituted by substituent group A, heterocyclyloxyoptionally substituted by substituent group A, carbocyclyl aminooptionally substituted by substituent group A or heterocyclylaminooptionally substituted by substituent group A;P^(R8) and P^(R9) may be taken together with an adjacent phosphorus atomto form heterocycle optionally substituted by substituent group A;P^(R10) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A,heterocyclyl group optionally substituted by substituent group A,carbocyclylalkyl optionally substituted by substituent group A orheterocyclylalkyl optionally substituted by substituent group A; andP^(R11) is alkyl optionally substituted by substituent group A, alkenyloptionally substituted by substituent group A, carbocyclyl groupoptionally substituted by substituent group A, or heterocyclyl groupoptionally substituted by substituent group A;Substituent group A; oxo, alkyl, hydroxyalkyl, amino, alkylamino,carbocyclyl, heterocyclyl, carbocyclylalkyl, alkylcarbonyl, halogen,hydroxy, carboxy, alkylcarbonylamino, alkylcarbonylaminoalkyl,alkylcarbonyloxy, alkyloxycarbonyl, aryloxycarbonylalkyl,alkyloxycarbonyloxy, alkylaminocarbonyloxy, alkylaminoalkyl, alkyloxy,cyano, nitro, azido, alkylsulfonyl, trialkylsilyl and phospho.

Examples of further preferred embodiment of the group to form a prodruginclude following groups.—C(═O)—P^(R0),  a)—C(═O)—P^(R1),  b)—C(═O)—O—P^(R2),  g)—C(═O)—N(—K)(P^(R2)),  h)—C(═O)—O-L-O—P^(R2),  i)—C(P^(R3))₂—O—C(═O)—P^(R4),  l)—C(P^(R3))₂—O—C(═O)—O—P^(R4),  m)—C(P^(R3))₂—OC(═O)—O-L-O—P^(R4),  o)—C(P^(R3))₂—P^(R6),  v)—C(P^(R3))₂—C(P^(R3))₂—C(═O)—O—P^(R2),  x)—C(P^(R3))₂—N(—K)—C(═O)—O—P^(R2), and  y)—P(═O)(—P^(R8))(—P^(R9)),  z)wherein L is straight or branched alkylene;K is hydrogen, or alkyl optionally substituted by substituent group A;P^(R0) is alkyl optionally substituted by substituent group A;P^(R1) is carbocyclyl group optionally substituted by substituent groupA, or heterocyclyl group optionally substituted by substituent group A;P^(R2) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A,heterocyclyl group optionally substituted by substituent group A,carbocyclylalkyl optionally substituted by substituent group A, orheterocyclylalkyl optionally substituted by substituent group A;P^(R3) is each independently hydrogen or alkyl;P^(R4) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A, orheterocyclyl group optionally substituted by substituent group A;P^(R6) is carbocyclyl group optionally substituted by substituent groupA, or heterocyclyl group optionally substituted by substituent group A;P^(R8) is alkyloxy optionally substituted by substituent group A;P^(R9) is alkyloxy optionally substituted by substituent group A,alkylamino optionally substituted by substituent group A, carbocyclyloxyoptionally substituted by substituent group A, heterocyclyloxyoptionally substituted by substituent group A, carbocyclylaminooptionally substituted by substituent group A or heterocyclylaminooptionally substituted by substituent group A; andP^(R8) and P^(R9) may be taken together with an adjacent phosphorus atomto form heterocycle optionally substituted by substituent group A;Substituent group A; oxo, alkyl, alkylamino, carbocyclyl, heterocyclyl,alkylcarbonyl, halogen, hydroxy, alkylcarbonylamino, alkylcarbonyloxy,alkyloxycarbonyl, aryloxycarbonylalkyl, alkylaminocarbonyloxy, alkyloxy,nitro, azido, alkylsulfonyl and trialkylsilyl.

Among the above group to form a prodrug, a) —C(═O)—P^(R0), b)—C(═O)—P^(R1), g) —C(═O)—OP^(R2), h) —C(═O)—N(—K)(P^(R2)), j)—C(═O)—O-L-O—P^(R2), l) —C(P^(R3))₂—O—C(═O)—P^(R4), m)—C(P^(R3))₂—O—C(═O)—O—P^(R4), o) —C(P^(R3))₂—O—C(═O)—O-L-O—P^(R4), x)—C(P^(R3))₂—C(P^(R3))₂—C(═O)—O—P^(R2), and y)—C(P^(R3))₂—N(—K)—C(═O)—O—P^(R2) are preferable.

In particular, a more preferable embodiment of the group to form aprodrug is the following group,—C(P^(R3))₂—O—C(═O)—O—P^(R4)  m)wherein P^(R3) is each independently hydrogen or alkyl; andP^(R4) is alkyl optionally substituted by substituent group A,carbocyclyl group optionally substituted by substituent group A, orheterocyclyl group optionally substituted by substituent group A;Substituent group A; oxo, alkyl, alkylamino, carbocyclyl, heterocyclyl,alkylcarbonyl, halogen, hydroxy, alkylcarbonylamino, alkylcarbonyloxy,alkyloxycarbonyl, aryloxycarbonylalkyl, alkylaminocarbonyloxy, alkyloxy,nitro, azido, alkylsulfonyl and trialkylsilyl.

An embodiment of P^(R3) is each independently hydrogen or alkyl, andpreferably hydrogen.

An embodiment of P^(R4) is alkyl optionally substituted by substituentgroup A, carbocyclyl group optionally substituted by substituent groupA, or heterocyclyl group optionally substituted by substituent group A,and preferably methyl, ethyl or the like.

An embodiment of Substituent group A includes oxo, alkyl, alkylamino,carbocyclyl, heterocyclyl, alkylcarbonyl, halogen, hydroxy,alkylcarbonylamino, alkylcarbonyloxy, alkyloxycarbonyl,aryloxycarbonylalkyl, alkylaminocarbonyloxy, alkyloxy, nitro, azido,alkylsulfonyl and trialkylsilyl, and preferably fluorine, chlorine,hydroxyl, methyl and ethyl.

Examples of another embodiment of a preferable substituent of the groupto form a prodrug include following groups.

Examples of another embodiment of a preferable substituent of the groupto form a prodrug include following groups.

Examples of an embodiment of a particularly preferable substituent ofthe group to form a prodrug include following groups.

(Method for Producing Compound of the Present Invention)

A general method for producing the compound used in the presentinvention will be exemplified below. As to the extraction andpurification, treatment which is performed in a normal experiment oforganic chemistry may be conducted.

Synthesis of the compound used in the present invention can be carriedout referring to the procedures known in the art.

As a raw material compound, commercially available compounds, compoundsdescribed in the present description, compounds described in thereferences cited in the present description, and other known compoundscan be utilized.

When one wants to obtain a salt of the compound of the presentinvention, in the case where the compound used in the present inventionis obtained in a form of a salt, it may be purified as it is and, in thecase where the compound used in the present invention is obtained in afree form, a salt may be formed by a normal method by dissolving orsuspending the compound in a suitable organic solvent, and adding anacid or a base.

In addition, the compound used in the present invention and apharmaceutically acceptable salt thereof are present in a form ofadducts with water or various solvents (hydrate or solvate) in somecases, and these adducts are used in the present invention.

In a general synthesis method as well as Examples and IntermediateSynthesis Examples, the meaning of each abbreviation is as follows.

Boc: tert-butoxycarbonyl

DBU: diazabicycloundecene

DMA: N,N-dimethylacetamide

DMF: N,N-dimethylformamide

HATU: O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate

NMP: N-methylpyrrolidone

OBn: benzyloxy

THF: tetrahydrofuran

T3P: propyl phosphonic anhydride

WSC.HCl: N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride

The up and down of the “wedge” and “broken line wedge” indicates theabsolute configuration.

(Preparation 1)

wherein P¹ is hydroxyl protective group) R^(P) is acetal protectivegroup) L is leaving group) Other each symbol is same as above.First Step

Compound A3 can be obtained by adding Compound A2 to Compound A1 in thepresence of a dehydration-condensation agent such asdicyclohexylcarbodiimide, carbonyldiimidazole,dicyclohexylcarbodiimide-N-hydroxybenzotriazole,4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride,hexafluorophosphoric acid2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium, WSC.HCl,HATU, etc. in a solvent such as DMF, THF, dichloromethane, acetonitrileetc. or in a mixed solvent thereof, and performing a reaction at −20° C.to 60° C., preferably −10° C. to 40° C. for 0.1 hours to 24 hours,preferably 1 hour to 12 hours.

Alternatively, Compound A3 can be obtained by adding an acylatingreagent such as diphenylchlorophosphate, thionyl chloride, oxalylchloride etc. to Compound A1 in the presence or absence of a base suchas pyridine, triethylamine, diisopropylethylamine, 1-methylimidazole,etc. in the presence of a solvent such as THF, dioxane, dichloromethane,DMF etc., thereby, generating acid chloride, and adding Compound A2having a substituent corresponding to an objective compound, andperforming a reaction at −20° C. to 60° C., preferably −10° C. to 40° C.for 0.1 hours to 24 hours, preferably 0.5 hours to 12 hours.

Second Step

Compound A4 can be obtained by adding potassium carbonate, sodiumcarbonate, and O-(2,4-dinitrophenyl)hydroxylamine to Compound A3 in thepresence of a solvent such as DMF, DMA, NMP, THF, etc., and performing areaction at 10° C. to 60° C., preferably 20° C. to 40° C. for 0.1 hoursto 48 hours, preferably 1 hour to 24 hours.

Third Step

A deprotecting reaction of an acetal protective group of Compound A4 canbe performed by the general method described in Protective Groups inOrganic Synthesis, Theodora W Green (John Wiley & Sons) etc. Thereafter,a generated aldehyde group is subjected to an intramolecular reaction,thereby, Compound A5 can be obtained.

For example, racemate of Compound A5 can be obtained by adding aceticacid and/or paratoluenesulfonic acid, methanesulfonic acid etc., toCompound A4 in the presence of a solvent such as DMF, toluene, THF,etc., and performing a reaction at 10° C. to 80° C., preferably 30° C.to 60° C. for 0.5 hours to 12 hours, preferably 1 hour to 6 hours.Compound A5 can be obtained by optical resolution of the racemate ofCompound A5 by SFC or HPLC (chiral column).

Fourth Step

Compound A7 can be obtained by adding Compound A6, and a base such assodium carbonate, potassium carbonate, cesium carbonate, etc. toCompound A5 in the presence of a solvent such as DMF, DMA, NMP, THF,etc. or in a mixed solvent thereof, and performing a reaction at 0° C.to 60° C., preferably 10° C. to 40° C. for 0.1 hours to 48 hours,preferably 1 hour to 24 hours.

Alternatively, Compound A7 can be obtained by adding Compound A6, andT3P, methane sulfonic acid or para-toluene sulfonic acid to Compound A5in the presence of a solvent such as DMF, ethyl acetate, butyl acetate,1,4-dioxane etc. or in a mixed solvent thereof, and performing areaction at 40° C. to 150° C., preferably 60° C. to 120° C. for 0.1hours to 48 hours, preferably 1 hour to 24 hours.

Fifth Step

A deprotecting reaction of hydroxyl protective group of Compound A7 canbe performed by the general method described in Protective Groups inOrganic Synthesis, Theodora W Green (John Wiley & Sons) etc.

Sixth Step

Compound (III) can be obtained by the general method includingconverting a hydroxyl group of Compound (II) into an ester group orether group.

For example, the method described in Protective Groups in OrganicSynthesis, Theodora W Green (John Wiley & Sons), Prog. Med. 5: 2157-2161(1985), and Supplied by The British Library—“The world's Knowledge”,etc. can be utilized.

(Preparation 2)

wherein P² is NH protective group; L¹ and L² is leaving group; Othereach symbol is same as above.First Step

Compound B2 can be obtained by adding Compound A2 and halogenated alkylsuch as methyl iodide to Compound B1 in the presence of a base such asdiazabicycloundecene in a solvent such as DMF, THF, dichloromethane,acetonitrile, etc. or in a mixed solvent thereof, and performing areaction at −20° C. to 60° C., preferably −10° C. to 40° C. for 0.1hours to 24 hours, preferably 1 hour to 24 hours.

Alternatively, Compound B2 can be obtained by adding acylating reagentsuch as diphenylchlorophosphate, thionyl chloride, oxalyl chloride, etc.to Compound B1 in a solvent such as THF, dioxane, dichloromethane, DMF,etc. or in a mixed solvent thereof, and adding alcohol in the presenceof a base such as pyridine, triethylamine, diisopropylethylamine,1-methylimidazole, etc., and performing a reaction at −20° C. to 60° C.,preferably −10° C. to 40° C. for 0.1 hours to 24 hours, preferably 0.5hours to 12 hours.

Second Step

Compound B3 can be obtained by adding para-toluene sulfonic acidpyridinium and hydrazine protected by Boc etc. to Compound B2 in asolvent such as THF, dioxane, dichloromethane, DMF etc., or in a mixedsolvent thereof, and performing a reaction at 10° C. to 150° C.,preferably 40° C. to 100° C. for 1 hour to 48 hours, preferably 1 hourto 24 hours.

Third Step

A deprotecting reaction of amino protective group Compound B3 can beperformed by the general method described in Protective Groups inOrganic Synthesis, Theodora W Green (John Wiley & Sons) etc.

Fourth Step

Compound B6 can be obtained by adding a base such as n-butyl lithium,etc. to Compound B5 in a solvent such as THF, dioxane, dichloromethane,DMF etc., or in a mixed solvent thereof, and then adding haloformic acidalkyl and performing a reaction for 0.1 hours to 48 hours, preferably 1hour to 24 hours.

Fifth Step

Compound B7 can be obtained by adding reducing agent such as Lithiumdiisobutylaluminum hydride, etc. to Compound B6 in a solvent such asTHF, dioxane, dichloromethane, DMF etc., or in a mixed solvent thereof,and performing a reaction for 0.1 hours to 48 hours, preferably 1 hourto 24 hours.

Sixth Step

Compound B8 can be obtained by adding para-toluene sulfonic acid ormethane sulfonic acid to Compound B7 in alcohol, and performing areaction at 0° C. to 100° C. for 0.1 hours to 48 hours, preferably 1hour to 24 hours.

Seventh Step

Compound B10 can be obtained by adding haloformic acid alkyl to CompoundB9 in the presence or absence of a base such as pyridine, triethylamine,diisopropylethylamine, 1-methylimidazole, etc., in a solvent such asTHF, dioxane, dichloromethane, DMF etc., or in a mixed solvent thereof,and performing a reaction at −40° C. to 40° C. for 0.1 hours to 48hours, preferably 1 hour to 24 hours.

Eighth Step

Compound B8 can be obtained by immersing carbon electrode (anode) andplatinum electrode (cathode) to Compound B10 in a solvent such asalcohol in the presence of a base such as potassium carbonate andtetraethylaminium perchlorate, and flushing with a constant current of0.1˜1.0 A with stirring for 0.1 hours to 48 hours, preferably 1 hour to24 hours.

Ninth to Tenth Step

Compound (I) can be obtained from Compound B4 and B8 in the same manneras in the third to sixth steps in preparation 1.

The compounds used in the present invention have cap-dependentendonuclease inhibitory activity and are useful as therapeutic orpreventive agents for influenza.

The compounds (parent compounds and/or prodrugs) used in the presentinvention are useful for symptoms and/or diseases which are induced byinfluenza virus. For example, they are useful for treating, preventing,and/or improving symptoms of, cold-like symptoms accompanying fever,chill, headache, muscular or joint pain, fatigue etc., airwayinflammation symptoms such as sore throat, nasal secretion, nasalcongestion, cough, sputum etc., gastrointestinal symptoms such asabdominal pain, vomitus, diarrhea etc. and, further, complicationsaccompanying secondary infection such as acute encephalopathy andpneumonia. That is to say, the compounds used in the present inventionare useful for treating and/or preventing influenza virus infectiousdiseases.

The compounds used in the present invention are effective for shorteningtime to alleviation of influenza symptoms. For example, they can shortenthe time to alleviation of influenza symptoms about 20 to 40 hours,preferably about 25 to 30 hours. Specifically, they can shorten thetimes until “cough”, “sore throat”, “headache”, “nasal congestion”,“feverishness or chills”, “muscular or joint pain”, and “fatigue” arealleviated. In particular, they are useful for shortening the timesuntil “nasal congestion”, “muscular or joint pain”, “fatigue”,“feverishness or chills”, and “headache” are alleviated. Further, theyare useful for shortening the times until “nasal congestion” and“muscular or joint pain” are alleviated.

Furthermore, since the compounds (parent compounds and/or prodrugs) usedin the present invention reduces the influenza virus in the body in ashort period of time, it can be an excellent pharmaceutical useful fortreating and/or preventing influenza virus infectious diseases. Afterthe administration of the compounds used in the present invention, theeffect of decreasing the influenza virus amount in the body is observedwithin 72 hours, preferably within 48 hours, more preferably within 24hours, and it is expected an earlier therapeutic effect is obtained ascompared with other drugs.

Moreover, the compounds used in the present invention have usefulness asmedicines.

For example, since the compounds (prodrugs) used in the presentinvention have advantages that oral absorbability is high, goodbioavailability is exhibited, good clearance is exhibited, and pulmonarytransitivity is high, they can be excellent medicaments.

Since the compounds (parent compounds) used in the present inventionhave the effects such as high inhibitory activity on capstructure-dependent endonuclease, and high selectivity due to avirus-specific enzyme, they can be medicaments having reduced sideeffects.

Further, the compounds (parent compounds and/or prodrugs) used in thepresent invention also have advantages that metabolism stability ishigh, solubility is high, oral absorbability is high, goodbioavailability is exhibited, good clearance is exhibited, pulmonarytransitivity is high, a half life is long, a non-protein binding rate ishigh, hERG channel inhibition is low, CYP inhibition is low, CPE(CytoPathic Effect) inhibiting effect is recognized, and/or negativeeffects are exhibited in a phototoxicity test, an Ames test and a genetoxicity test, or toxicity such as liver damage is not caused.Therefore, the pharmaceutical composition of the present invention canbe an excellent medicament.

The compounds (parent compounds and/or prodrugs) used in the presentinvention can be administered orally or parenterally. In the case oforal administration, the compounds used in the present invention can bealso used as a normal preparation, for example, as any dosage form ofsolid preparations such as tablets, powders, granules, capsules etc.;solutions; oleaginous suspensions; or liquid preparations such as syrupsor elixirs etc. In the case of parenteral administration, the compoundsused in the present invention can be used as aqueous or oleaginoussuspension injectables, or nose drops. Upon preparation of them,conventional excipients, binders, lubricants, aqueous solvents,oleaginous solvents, emulsifiers, suspending agents, preservatives,stabilizers etc. can be arbitrarily used. The pharmaceutical compositionof the present invention can be produced by combining (for example,mixing) a therapeutically and/or prophylactically effective amount ofthe compound used in the present invention with pharmaceuticallyacceptable carriers or diluents. The compounds used in the presentinvention can be suitably used as oral preparations because of theirhigh oral absorbability.

A dose of the compounds used in the present invention is differentdepending on an administration method, an age, a weight and the state ofa patient, and a kind of a disease and, usually, in the case of oraladministration, about 0.05 mg to 3000 mg, preferably about 0.1 mg to1000 mg, more preferably about 10 mg to 80 mg, particularly preferablyabout 10 to 40 mg for adult per day may be administered, if necessary,in divided doses. In another embodiment, in the case of adults, about 40mg or 80 mg may be administered in a single dose. In the case ofchildren, about 5 to 40 mg may be administered in a single dosedepending on the body weight. In addition, in the case of parenteraladministration, about 0.01 mg to 1000 mg, preferably about 0.05 mg to500 mg, or about 1 mg to 80 mg for adult per day is administered. Thedose may be administered once daily or may be divided into multipledoses per day.

The compounds used in the present invention can be used in combinationwith other drugs or the like (hereinafter referred to as combinationdrugs) to increase the activity of the compounds, reduce the dose of thecompounds, or the like. In the case of treating influenza, the compoundscan be used combined with or in a coupled formulation with neuraminidaseinhibitor (e.g., Oseltamivir, Zanamivir, Peramivir, Inabiru and thelike); RNA-dependent. RNA polymerase inhibitor (e.g., Favipiravir); M2protein inhibitor (e.g., Amantadine); PB2 Cap binding inhibitor (e.g.,VX-787); anti-HA antibody (e.g., MHAA4549A); Immune agonists (e.g.,Nitazoxanide) are also possible. In this case, the timing ofadministration for a compound used in the present invention and thecombination drug is not limited. They can be administered to thesubjects to be treated, at a time or at different times. Furthermore,the combination drug with a compound used in the present invention canbe administered as two or more formulations independently comprisingeach active ingredient or a single formulation comprising each activeingredient.

The dose for combination drugs may be appropriately selected inreference to the clinical dose. The compounding ratio of the compoundsused in the present invention and co-administered drugs may beappropriately selected depending on the subject to be treated,administration route, disease to be treated, symptoms, combination ofthe drugs and the like. For administration in humans, for example, 1part by weight of the compounds used in the present invention may beused in combination with 0.01 to 100 parts by weight of co-administereddrugs.

The present invention will be explained in more detail below by way ofExamples, Intermediate Synthesis Examples, as well as Test Examples ofthe present invention, but the present invention is not limited to them.

The NMR analysis obtained in each example was carried out in 300 MHz,and was measured using DMSO-d₆, CDCl₃.

The term RT represents a retention time at LC/MS: liquidchromatography/mass spectrometry, and was measured under the followingconditions.

(Measurement Conditions)

(1) Column: ACQUITY UPLC (Registered trademark) BEH C18 (1.7 μm i.d.2.1×50 mm) (Waters)

Flow rate: 0.8 mL/min

UV detection wavelength: 254 nm

Mobile phase: [A]: a 0.1% formic acid-containing aqueous solution, [B]:a 0.1% formic acid-containing acetonitrile solution

Gradient: a linear gradient of 5% to 100% solvent [B] was carried out in3.5 minutes, and 100% solvent [B] was kept for 0.5 minutes.

(2) Column: Shim-pack XR-ODS (2.2 μm, i.d. 50×3.0 mm) (Shimadzu)

Flow rate: 1.6 mL/min

UV detection wavelength: 254 nm

Mobile phase: [A]: a 0.1% formic acid-containing aqueous solution, [B]:a 0.1% formic acid-containing acetonitrile solution

Gradient: a linear gradient of 10% to 100% solvent [B] was carried outin 3 minutes, and 100% solvent [B] was kept for 0.5 minutes.

Measurement of Powder X-Ray Diffraction Pattern

The powder X-ray diffraction of crystals obtained in each example wasmeasured in accordance with X-ray powder diffraction method described inthe General Tests, Processes and Apparatus of the JapanesePharmacopoeia. Measurement conditions are shown below.

(Apparatus)

MinFlex 600 RINT-TTR III manufactured by Rigaku Corporation

(Operation Method)

Detector: High-speed 1-dimensional detector (D/Tec Ultra 2) and variableknife edge

Measurement method: Reflection method

Type of light source: Cu tube

Wavelength used: CuKα radiation

Tube current: 10 mA or 15 mA

Tube voltage: 30 Kv or 40 Kv

Sample plate: Aluminum or glass

X-ray incidence angle (θ): 3-40°, sampling width: 0.01° or

X-ray incidence angle (θ): 4-40°, sampling width: 0.02°

In general, the diffraction angle (2θ) in powder X-ray diffraction mayhave a margin of error within the range of ±0.2°, and therefore thevalue of the diffraction angle also encompasses about ±0.2° range of thenumerical value. Accordingly, the present invention encompasses not onlya crystal, the peak diffraction angles of which in powder X-raydiffraction completely match, but also a crystal, the peak diffractionangles of which match within a margin of error of about ±0.2°.

Example 1-1: Method for Producing Compound i1

First Step

To a solution of Compound 1 (5.0 g, 49.5 mmol) in THF (100 mL) was addeddropwise 1.62 mol/L n-butyllithium in hexane (90.5 mL, 49.5 mmol) at−78° C. under a nitrogen atmosphere, and the mixture was stirred at −78°C. for 2 hours. A solution of chloroformate allyl (5.96 g, 49.5 mmol) inTHF (20 mL) was added dropwise thereto, and the mixture was stirred at−78° C. for 2 hours. The mixture was quenched with a saturated aqueoussolution of ammonium chloride, warmed up to room temperature, andextracted with ethyl acetate. The obtained organic layer was washed withbrine, dried over anhydrous magnesium sulfate, and concentrated underreduced pressure to obtain Compound 2 (5.66 g, 62%).

1H-NMR (CDCl3) δ:3.83 (t, J=8.0 Hz, 2H), 3.92 (t, J=8.0 Hz, 2H), 4.26(s, 2H), 4.78 (d, J=8.0 Hz, 2H), 5.30 (d, J=12.0 Hz, 1H), 5.44 (d,J=16.0 Hz, 1H), 5.93-6.03 (m, 1H),

Second Step

To a solution of Compound 2 (6.6 g, 35.6 mmol) in THF (66 mL) was addeddropwise 1.03 mol/L DIBAL-H in hexane (45.0 mL, 46.3 mmol), and themixture was stirred at −78° C. for 1 hour. The mixture was quenched withacetone, an aqueous solution of Rochelle salt was added thereto. Themixture was stirred, and extracted with ethyl acetate. The obtainedorganic layer was washed with brine, dried over anhydrous magnesiumsulfate, and concentrated under reduced pressure to obtain Compound 3(6.21 g, 93%).

1H-NMR (CDCl3) δ: 3.44 (br, 1H), 3.50-3.64 (m, 2H), 3.71 (br, 1H), 3.95(d, J=8.0 Hz, 2H), 4.64 (d, J=8.0 Hz, 2H), 5.24 (d, J=12.0 Hz, 1H), 5.40(d, J=16.0 Hz, 1H), 5.47 (d, J=4 Hz, 1H), 5.87-6.00 (m, 1H)

Third Step

To a solution of Compound 3 (6.2 g, 33.1 mmol) in methanol (65 mL) wasadded p-Toluenesulfonic acid monohydrate (0.63 g, 3.31 mmol), and themixture was stirred at room temperature over night. The mixture wasquenched with an aqueous solution of sodium hydrogen carbonate,concentrated, and extracted with ethyl acetate. The obtained organiclayer was washed with brine, dried over anhydrous magnesium sulfate, andconcentrated under reduced pressure to obtain Compound 4 (5.77 g, 87%).

1H-NMR (CDCl3) δ:3.34 (s, 3H), 3.55 (br, 2H), 3.73-3.99 (m, 3H), 4.64(d, J=8.0 Hz, 2H), 5.10-5.20 (m, 1H), 5.25 (d, J=8.0 Hz, 1H), 5.33 (d,J=16 Hz, 1H), 5.88-6.05 (m, 1H)

Fourth Step

To a solution of Compound 5 (20.0 g, 81 mmol) in DMF (100 mL) were addedethyl iodide (22.8 g, 146 mmol) and diazabicycloundecene (18.4 mL, 122mmol), and the mixture was stirred at room temperature over night. Themixture was poured into 10% aqueous solution of ammonium chloride, andextracted with ethyl acetate. The obtained organic layer was washed withbrine, dried over anhydrous magnesium sulfate, and concentrated underreduced pressure to obtain Compound 6 (22.3 g, 100%).

1H-NMR (CDCl3) δ: 1.23 (t, J=8.0 Hz, 3H), 4.28 (q, J=8.0 Hz, 2H), 5.16(s, 2H), 6.57 (d, J=4.0 Hz, 1H), 7.28-7.48 (m, 5H), 8.21 (d, J=4.0 Hz,1H).

Fifth Step

To a solution of Compound 6 (500 mg, 1.82 mmol) in DMA (5.0 mL) wereadded pyridinium p-toluenesulfonate (1.37 g, 5.47 mmol) andBoc-hydrazine (361 mg, 2.74 mmol), and the mixture was stirred at 60° C.for 14 hours. To the mixture was added water and the mixture wasextracted with ethyl acetate. The obtained organic layer was washed witha saturated aqueous solution of ammonium chloride and brine, dried overanhydrous magnesium sulfate, and concentrated under reduced pressure.The obtained residue was purified by silica gel column chromatography(chloroform-methanol) to obtain Compound 7 (519 mg, 73%).

1H-NMR (CDCl3) δ:1.24 (t, J=8.0 Hz, 3H), 1.46 (s, 9H), 4.26 (q, J=8.0Hz, 2H), 5.28 (s, 2H), 6.40 (d, J=8.0 Hz, 1H), 7.27-7.38 (m, 4H),7.40-7.45 (m, 2H).

Sixth Step

Compound 7 (500 mg, 1.29 mmol) was dissolved in 4 mol/L hydrogenchloride in ethyl acetate (5 mL), and the mixture was stirred at roomtemperature for 1 hour. The mixture was concentrated under reducedpressure. To the obtained residue was added a saturated aqueous solutionof sodium hydrogen carbonate, and the mixture was extracted withdichloromethane. The obtained organic layer was washed with brine, driedover anhydrous magnesium sulfate, and concentrated under reducedpressure to obtain Compound 8 (369 mg, 99%).

1H-NMR (CDCl3) δ: 1.26 (t, J=8.0 Hz, 3H), 4.31 (q, J=8.0 Hz, 2H), 5.24(s, 2H), 6.47 (d, J=8.0, 1H), 7.28-7.44 (m, 5H), 7.64 (d, J=8.0, 1H).

Seventh Step

To a solution of Compound 8 (365 mg, 1.27 mmol) and Compound 4 (306 mg,1.52 mmol) in acetonitrile (8 mL) was added dropwise tin chloride (0.223mL, 1.90 mmol) at −25° C. under a nitrogen atmosphere, and the mixturewas stirred at −25° C. for 45 minutes. The mixture was quenched with asaturated aqueous solution of sodium hydrogen carbonate, anddichloromethane was added thereto. The mixture was stirred at roomtemperature and filtered through Celite, and filtrate was extracted withdichloromethane. The obtained organic layer was washed with brine, driedover anhydrous magnesium sulfate, and concentrated under reducedpressure to obtain crude Compound 9. The obtained Compound 9 wasdissolved in THF (8 mL), morpholine (1.10 mL, 12.7 mmol) andtetrakistriphenylphosphinepalladium (146 mg, 0.127 mmol) were addedthereto, and the mixture was stirred at room temperature for 2 hours. Tothe mixture was added diethyl ether (16 mL), and the precipitated solidwas filtered and dried to obtain Compound 10 (418 mg, 100%). 1H-NMR(CDCl3) δ: 2.90-2.99 (m, 1H), 3.13 (t, J=12.0 Hz, 1H), 3.40-3.46 (m,1H), 4.00-4.08 (m, 1H), 4.14 (d, J=12.0 Hz, 1H), 5.07 (s, 2H), 6.22 (d,J=8.0 Hz, 1H), 7.29-7.40 (m, 3H), 7.56 (d, J=8.0 Hz, 2H), 7.71 (d, J=8.0Hz, 1H)

Eighth Step

To a suspension of (R)-2-Tetrahydrofurioic Acid (855 mg, 7.36 mmol) andCompound 10 (2.00 g, 6.11 mmol) in ethyl acetate (9 ml) were addedpyridine (4.00 ml, 49.6 mmol) and T3P (50% in ethyl acetate, 11.0 ml,18.5 mmol) at room temperature, and the mixture was stirred over night.The precipitated solid was filtered and washed with ethyl acetate (4 ml)and ethanol (4 ml). The obtained solid was suspended in ethanol (6 ml)and the suspension was stirred at room temperature for 6.5 hours. Thesuspension was filtered and the obtained solid was washed with ethanol(2 ml) twice to obtain Compound 11 (1.18 g, 45.4%).

¹H-NMR (DMSO) δ: 1.80-1.94 (m, 2H), 1.95-2.14 (m, 2H), 3.21-3.35-(m,2H), 3.50-3.60 (m, 1H), 3.70-3.82 (m, 3H), 4.00-4.05 (m, 1H), 4.32-4.38(m, 1H), 5.14 (dd, J=10.8 Hz, 21.6 Hz, 2H), 5.76-5.81 (m, 1H), 6.29 (d;J=4.8 Hz, 1H), 7.28-7.39 (m, 3H), 7.48-7.54 (m, 2H), 7.64-7.75 (m, 1H)

Ninth Step

To a suspension of Compound 11 (500 mg, 1.18 mmol) in ethanol (3.5 ml)was added DBU (0.0035 ml, 0.023 mmol) at room temperature, and themixture was stirred for 30 minutes. To the obtained suspension was addeddiisopropylether (6.5 ml), and the mixture was stirred at roomtemperature for 30 minutes. The precipitated solid was filtered andwashed with ethyl acetate (1.5 ml) twice to obtain Compound i1 (346 mg,89.9%).

¹H-NMR (DMSO) δ: 2.80-3.00 (m, 1H), 3.10-3.18 (m, 1H), 3.38-3.50 (m,1H), 3.98-4.08 (m, 2H), 4.10-4.20 (m, 1H), 4.76-4.84 (m, 1H), 5.04-5.14(m, 2H), 6.22 (m, J=7.6 Hz, 1H), 7.27-7.40 (m, 4H), 7.56-7.60 (m, 2H),7.70 (d, J=7.6 Hz, 1H)

Example 1-2: Method for Producing Compound i2

First Step

To a suspension of Compound 13 (8.0 g, 50.8 mmol) in dichloromethane(120 mL) was added triethylamine (17.6 mL, 127 mmol) under ice-waterbath, and allyl chloroformate (6.44 mL, 60.9 mmol) was added dropwisethereto, and the mixture was stirred at 0° C. for 1 hour. To the mixturewas added water, and the mixture was extracted with dichloromethane. Theobtained organic layer was washed with 5% aqueous solution of citricacid and a saturated aqueous solution of sodium hydrogen carbonate,dried over anhydrous magnesium sulfate, and concentrated under reducedpressure to obtain Compound 14 (10.1 g, 97%).

1H-NMR (CDCl3) δ: 1.96 (br, 4H), 3.62 (s, 4H), 4.60 (s, 2H), 5.22 (d,J=12.0 Hz, 1H), 6.30 (d, J=16.0 Hz, 1H), 5.86-5.99 (m, 1H)

Second Step

To a solution of Compound 14 (0.9 g, 4.39 mmol), potassium carbonate (60mg, 0.44 mmol) and tetraethylammonium perchlorate (50 mg, 0.22 mmol) inmethanol (30 mL) were immersed carbon electrode (anode) and platinumelectrode (cathode), and the mixture was flushed with a constant currentof 0.1 A with stirring at room temperature for 6 hours. To the mixturewere added ethyl acetate and water, and the mixture was extracted withethyl acetate. The obtained organic layer was dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure to obtainCompound 15 (992 mg, 96%).

1H-NMR (CDCl3) δ: 1.81-2.15 (m, 3H), 2.39 (t, J=12.0 Hz, 1H), 3.27 (s,3H), 3.61 (s, 1H), 4.11 (br, 1H), 4.61 (br, 2H), 5.20-5.36 (m, 2H), 5.57(br, 1H), 5.88-5.99 (m, 1H)

Third Step

Compound 16 was obtained in the same manner as in the seventh and eighthsteps in example 1-1.

Fourth Step

The optical resolution of Compound 16 (870 mg, 2.41 mmol) by WatersSFC30 System (Daicel CHIRALPAK IB, liquefied carbon dioxide-methanol)gave Compound i2 (270 mg, 31%).

Analysis Condition

<Waters SFC30 System>

Column: CHIRALPAK IB/SFC (5 μm, i.d. 250×4.6 mm) (DAICEL)

Flow rate: 8.0 mL/min; UV detection wavelength: 254 nm

Back pressure: 100 bar

Mobile phase: [A]: liquefied carbon dioxide, [B]: methanol

Gradient: 5% solvent [B] was kept for 1 minute, a linear gradient of 5%to 40% solvent [B] was carried out in 6 minutes, 40% solvent [B] waskept for 2 minutes, and 5% solvent [B] was kept for 1 minute.

Elution time: 7.3 minutes

Example 1-3: Method for Producing Compound i3

First Step

To a solution of Compound 17 (4.00 g, 16.3 mmol) in dichloromethane (40mL) were added oxalyl dichloride (1.56 mL, 17.9 mmol) and DMF (0.013 mL,0.162 mmol) under iced-bath, and the mixture was warmed up to roomtemperature and stirred for 5 hours. The mixture was concentrated underreduced pressure, and the obtained residue was dissolved indichloromethane (40 mL), 2,2,2-trifluoroethanol (2.44 g, 24.4 mmol),triethylamine (4.50 mL, 32.5 mmol) and 4-(dimethylamino)pyridine (99.0mg, 0.812 mmol) were added thereto under iced-bath, and the mixture waswarmed up to room temperature and stirred for 1 hour. The mixture wasconcentrated under reduced pressure and to the obtained residue wasadded 1 mol/L aqueous solution of hydrochloric acid, and the mixture wasextracted with ethyl acetate. The obtained organic layer was washed with1 mol/L aqueous solution of hydrochloric acid and brine, dried overanhydrous magnesium sulfate to obtain Compound 18 (5.33 g, 100%).

1H-NMR (CDCl3) δ: 4.64 (q, J=8.2 Hz, 2H), 5.38 (s, 2H), 6.49 (d, J=5.6Hz, 1H), 7.30-7.38 (m, 3H), 7.43-7.49 (m, 2H), 7.75 (d, J=5.6 Hz, 1H).

Second and Third Steps

Compound 20 was obtained in the same manner as in the fifth and sixthsteps in example 1-1.

1H-NMR (CDCl3) δ: 4.55 (q, J=8.3 Hz, 2H), 5.18 (s, 2H), 5.29 (s, 2H),6.37 (d, J=7.8 Hz, 1H), 7.30-7.42 (m, 6H).

Fourth and Fifth Steps

Compound 23 was obtained in the same manner as in the seventh step inexample 1-1.

LC/MS (ESI): m/z=342.1 [M+H]⁺, RT=1.00, 1.09 min, method (1)

Sixth Step

To a solution of Compound 23 (820 mg, 2.40 mmol) in dichloromethane(16.5 mL) were added Boc₂O (0.837 mL, 3.60 mmol), triethylamine (0.499mL, 3.60 mmol) and 4-(dimethylamino)pyridine (44.0 mg, 0.360 mmol), andthe mixture was stirred at room temperature for 3.5 hours. To themixture was added 1 mol/L aqueous solution of hydrochloric acid and themixture was extracted with ethyl acetate. The obtained organic layer waswashed with 1 mol/L aqueous solution of hydrochloric acid and brine,dried over anhydrous sodium sulfate, and concentrated under reducedpressure. The obtained residue was purified by silica gel columnchromatography (chloroform-methanol) to obtain Compound 24 (593 mg, 56%)and Compound i3 (170 mg, 16%).

Compound 24: LC/MS (ESI): m/z=441.9 [M+H]⁺, RT=1.67 min, method (1)

Seventh Step

Compound 24 (547 mg, 1.24 mmol) was dissolved in acetic acid (5.5 mL)and the mixture was stirred at 80° C. for 5 hours. The mixture wasconcentrated under reduced pressure and the obtained residue waspurified by silica gel column chromatography (chloroform-methanol) toobtain Compound i3 (454 mg, 100%). 1H-NMR (CDCl3) δ: 1.46 (d, J=6.4 Hz,3H), 3.45 (dd, J=10.5, 10.5 Hz, 1H), 3.55 (dd, J=11.7, 4.3 Hz, 1H), 3.92(dd, J=11.7, 3.6 Hz, 1H), 3.95-4.01 (m, 2H), 4.76 (dq, J=13.9, 4.3 Hz,1H), 5.19 (d, J=10.2 Hz, 1H), 5.22 (d, J=10.2 Hz, 1H), 5.36 (d, J=12.9Hz, 1H), 6.28 (d, J=7.8 Hz, 1H), 7.25 (d, J=7.8 Hz, 1H), 7.28-7.36 (m,3H), 7.56-7.61 (m, 2H).

Example 1-4: Method for Producing Compound III-2

First Step

Compound i1 (1100 g, 3360 mmol) and7,8-difluoro-6,11-dihydrodibenzothiepine-11-ol (977 g, 3697 mmol) weresuspended in 50 wt % T3P in ethyl acetate (3208 g, 5041 mmol) and ethylacetate (1.1 L). To the mixture was added methanesulfonic acid (436 ml,6721 mmol) at room temperature and the mixture was stirred at 70° C. for5.5 hours. To the mixture was added water under ice-water bath and themixture was stirred at room temperature for 1 hour. THF was addedthereto and the mixture was extracted with ethyl acetate. The obtainedorganic layer was washed with water and 8% aqueous solution of sodiumhydrogen carbonate, dried over anhydrous sodium sulfate, andconcentrated under reduced pressure. The obtained residue was dissolvedin THF (5.5 L) and potassium carbonate (790 g, 5713 mmol) was addedthereto. The mixture was warmed up to 50° C., benzyl bromide (240 ml,2016 mmol) was added dropwise thereto, and the mixture was stirred al60° C. for 8.5 hours. To the mixture was added dropwise 2 mol/L aqueoussolution of hydrochloric acid under ice-water bath, and the mixture wasstirred at room temperature for 10 minutes and extracted with ethylacetate. The obtained organic layer was washed with water and 8% aqueoussolution of sodium hydrogen carbonate and dried over anhydrous magnesiumsulfate. An activated carbon (Norit SX-2, 240 g) was added thereto, themixture was filtered through Celite, and the filtrate was concentratedunder reduced pressure. To the obtained residue was added ethyl acetateand hexane and the precipitated solid was filtered to obtain Compound 25(1019 g, 1776 mmol, 53%).

¹H-NMR (CDCl₃) δ: 2.88 (1H, t, J=11.2 Hz), 3.28-3.39 (2H, m), 3.72 (1H,d, J=12.6 Hz), 3.86 (1H, d, J=9.6 Hz), 4.03 (1H, d, J=13.9 Hz), 4.45(1H, d, J=8.6 Hz), 4.67 (1H, d, J=13.1 Hz), 5.19-5.26 (2H, m), 5.45 (1H,d, J=10.9 Hz), 5.63 (1H, d, J=10.9 Hz), 5.77 (1H, d, J=7.6 Hz), 6.40(1H, d, J=7.8 Hz), 6.68 (1H, t, J=6.9 Hz), 6.94-7.01 (2H, m), 7.03-7.12(3H, m), 7.29-7.38 (3H, m), 7.61 (2H, d, J=7.1 Hz).

Second Step

To a solution of Compound 25 (1200 g, 2092 mmol) in DMA (3.6 L) wasadded lithium chloride (443 g, 10.5 mol) at room temperature, and themixture was stirred at 80° C. for 3 hours. To the mixture were addedacetone (1.2 L), 0.5 mol/L aqueous solution of hydrochloric acid (6.0 L)and water (2.4 L) under ice-water bath, and the mixture was stirred for1 hour. The precipitated solid was filtered. The obtained solid wasdissolved in chloroform, isopropyl ether was added thereto, and theprecipitated solid was filtered to obtain Compound III-2 (950 g, 1965mmol, 94%).

¹H-NMR (CDCl₃) δ: 2.99 (1H, dt, J=17.5, 6.8 Hz), 3.47 (1H, td, J=11.9,2.5 Hz), 3.60 (1H, t, J=10.6 Hz), 3.81 (1H, dd, J=11.9, 3.3 Hz), 3.96(1H, dd, J=11.0, 2.9 Hz), 4.07 (1H, d, J=13.8 Hz), 4.58 (1H, dd, J=10.0,2.9 Hz), 4.67 (1H, dd, J=13.5, 1.9 Hz), 5.26-5.30 (2H, m), 5.75 (1H, d,J=7.8 Hz), 6.69 (1H, d, J=7.7 Hz), 6.83-6.87 (1H, m). 6.99-7.04 (2H, m).7.07-7.15 (3H, m).

Example 2: Method for Producing Compound III-42

First Step

Compound 34 (947 mg, 5.56 mmol) was dissolved in toluene (8 ml),triethylamine (0.848 ml, 6.12 mmol) was added thereto, and the mixturewas stirred at room temperature for 30 minutes. Diphenylphosphonoazide(1.32 ml, 6.12 mmol) was added thereto, the mixture was stirred at 80°C. for 1 hour, (9H-fluoren-9-yl) methanol (5.46 g, 27.8 mmol) was addedthereto, and the mixture was heated to reflux at 120° C. for 1 hour. Themixture was cooled to room temperature, and a saturated aqueous solutionof sodium hydrogen carbonate was added thereto to stop the reaction. Thereaction solution was extracted with ethyl acetate, and the obtainedorganic layer was washed with brine, dried over anhydrous sodiumsulfate, and concentrated under reduced pressure. The obtained residuewas purified by silica gel column chromatography (ethyl acetate-hexane)to obtain Compound 35 (1.5 g, 74%).

1H-NMR (CDCl3) δ:7.77 (2H, d, J=7.3 Hz), 7.60 (2H, d, J=7.3 Hz),7.41-7.39 (2H, m), 7.32-7.30 (2H, m), 5.10-5.07 (1H, m), 4.75 (1H, brs),4.41 (2H, d, J=6.6 Hz), 4.22 (1H, t, J=6.6 Hz), 3.15-3.12 (1H, m),3.04-3.01 (1H, m), 2.04-1.96 (2H, m), 1.68 (3H, s), 1.62 (3H, s),1.39-1.37 (1H, m), 1.17-1.16 (1H, m), 0.90 (3H, d, J=6.6 Hz), 0.87-0.83(1H, m)

Second Step

Compound 35 (204 mg, 0.561 mmol) was dissolved in a mixed solution ofdioxane (3 ml) and water (1.5 ml), then potassium osmate(VI) dihydrate(10.3 mg, 0.028 mmol) and sodium periodate (360 mg, 1.68 mmol) wereadded thereto, and the mixture was stirred at room temperatureovernight. The reaction was stopped with a 10% aqueous solution ofsodium thiosulphate, the reaction solution was extracted with ethylacetate, and the obtained organic layer was washed with brine, driedover anhydrous sodium sulfate, and concentrated under reduced pressureto obtain a crude product. The crude product was dissolved in methanol(4 ml), tosic acid monohydrate (13.8 mg, 0.072 mmol) was added thereto,and the mixture was stirred at room temperature for 2 hours. Thereaction was stopped by adding a saturated aqueous solution of sodiumhydrogen carbonate, the reaction solution was extracted with ethylacetate, and the obtained organic layer was washed with brine, driedover anhydrous sodium sulfate, and concentrated under reduced pressure.The obtained residue was purified by silica gel column chromatography(hexane-ethyl acetate) to obtain a diastereomeric mixture of Compound 36(115 mg, 58%).

1H-NMR (CDCl3) δ:7.80-7.73 (2H, m), 7.60-7.54 (2H, m), 7.45-7.28 (4H,m), 5.36 (0.5H, s), 4.95 (0.5H, s), 4.62-4.55 (1H, m), 4.48 (1H, d,J=6.6 Hz), 4.30-4.20 (1H, m), 3.90-3.82 (0.5H, m), 3.74-3.65 (m, 0.5H),3.20 (1.5H, s), 2.90 (1.5H, s), 2.57 (0.5H, t, J=11.4 Hz), 2.47 (0.5H,t, J=11.4 Hz), 1.90-1.70 (1H, m), 1.60-1.30 (2.5H, m), 0.93-0.80 (3.5H,m)

Third Step

Compound 36 (115 mg, 0.33 mmol) and methyl1-amino-3-(benzyloxy)-4-oxo-1,4-dihydropyridine-2-carboxylate (50 mg,0.182 mmol) were dissolved in acetonitrile (3 ml), and the mixture wascooled to −30° C. Tin tetrachloride (0.032 ml, 0.27 mmol) was addedthereto, and the mixture was stirred for 4 hours. Saturated sodiumhydrogen carbonate was added to the reaction solution, and the reactionsolution was extracted with methylene chloride. The obtained organiclayer was washed with a saturated aqueous solution of sodium chloride,dried over sodium sulfate, filtered, and concentrated under reducedpressure. The obtained residue was purified by silica gel columnchromatography (chloroform-methanol) to obtain a diastereomeric mixtureof Compound 37 (115 mg, 59%).

LC/MS (ESI): m/z=595 [M+H]⁺, RT=2.49, 2.63 min, method (1)

Fourth Step

Compound 37 (20.5 g, 34.5 mmol) was dissolved in THF (400 ml),piperidine (68.4 ml, 691 mmol) was added thereto, and the mixture wasstirred at room temperature for 2 hours. The reaction solution wasdiluted with diethyl ether (500 ml), and the produced precipitates werefiltered to obtain a crude product.

The crude product was dissolved in ethanol (200 ml), DBU (5.06 ml, 33.6mmol) was added thereto, and the mixture was stirred at 80° C. for 2hours. The reaction solution was concentrated, and the obtained solidswere recrystallized with THF to obtain Compound 38 (6.5 g, 57%).

LC/MS (ESI): m/z=340 [M+H]⁺, RT=1.29 min, method (1)

Fifth Step

Compound 38 (2.0 g, 5.89 mmol) and7,8-difluoro-6,11-dihydrodibenzo[b,e]thiepin-11-ol (2.34 g, 8.84 mmol)were dissolved in a solution of T3P in ethyl acetate (18 mL), and themixture was stirred in a sealed tube at 110° C. for 1.5 hours. Thereaction was stopped by adding water, the reaction solution wasextracted with ethyl acetate, and the obtained organic layer was washedwith brine, dried over anhydrous sodium sulfate, and concentrated underreduced pressure. The obtained residue was recrystallized withchloroform-hexane to obtain Compound 39 (1.1 g, 32%).

LC/MS (ESI): m/z=586 [M+H]⁺, RT=2.46 min, method (1)

Sixth Step

Compound 39 (1.1 g, 1.89 mmol) was dissolved in dimethylacetamide (10ml), lithium chloride (398 mg, 9.39 mmol) was added thereto, and themixture was stirred at 120° C. The reaction solution was diluted withethyl acetate, and the obtained organic layer was washed with 2 mol/Lhydrochloric acid, dried over anhydrous sodium sulfate, and concentratedunder reduced pressure to obtain Compound III-42 (556 mg, 59.6%).

1H-NMR (CDCl3) δ: 7.15-7.03 (4H, m), 7.01-6.94 (1H, m), 6.86-6.82 (1H,m), 6.68 (1H, d, J=7.8 Hz), 5.78 (1H, d, J=7.6 Hz), 5.35 (1H, dd,J=13.8, 2.4 Hz), 5.22 (1H, s), 4.65-4.57 (1H, m), 4.25 (1H, dd, J=11.4,2.5 Hz), 4.05 (1H, d, J=13.9 Hz), 2.18 (1H, t, J=12.4 Hz), 1.96 (1H, d,J=13.6 Hz), 1.87-1.57 (5H, m), 1.29-1.22 (2H, m), 0.91 (3H, d, J=6.6Hz).

LC/MS (ESI): m/z=497 [M+H]⁺, RT=2.16 min, method (1)

Example 3: Method for Producing Compound 42

First Step

Compound 40 (1.00 g, 13.3 mmol) was dissolved in THF (100 mL), 60%sodium hydride (0.69 g, 14.7 mmol) was added thereto at roomtemperature, and the mixture was stirred under a nitrogen stream at roomtemperature for 30 minutes. Ethyl chloroacetate (1.4 mL, 13.3 mmol) wasadded thereto, and the mixture was stirred under a nitrogen stream atroom temperature for 30 minutes and at 90° C. for 3 hours. Afterconcentration under reduced pressure, THF (40 mL) was added to theresidue, further, 60% sodium hydride (0.59 g, 14.7 mmol) was addedthereto, and the mixture was stirred under a nitrogen stream at roomtemperature for 30 minutes. Allyl chloroformate was added dropwisethereto, and the mixture was stirred at room temperature for 3 hours. Asaturated aqueous solution of ammonium chloride (30 mL) was addedthereto, and the reaction solution was extracted with ethyl acetate (150mL). The obtained organic layer was washed with water (50 mL) and brine(100 mL) and dried over anhydrous magnesium sulfate, and the obtainedresidue was purified by silica gel column chromatography (hexane-ethylacetate) to obtain Compound 41 (0.96 g, 36%).

¹H-NMR (CDCl₃) δ: 1.42 (d, J=6.5 Hz, 3H), 3.77-3.89 (m, 2H), 4.19 (d,J=17.4 Hz, 1H), 4.27-4.36 (m, 2H), 4.74-4.83 (m, 2H), 5.31 (dd, J=10.4,1.4 Hz, 1H), 5.46 (dd, J=17.2, 1.4 Hz, 1H), 5.98 (dddd, J=17.2, 10.4,5.6, 5.6 Hz, 1H).

LC/MS (ESI): m/z=199.8 [M+H]⁺, method (1)

Second Step

Compound 41 (2.69 g, 13.5 mmol) was dissolved in THF (30 mL) under anitrogen atmosphere and cooled to −78° C. with dry ice-acetone. A 1.02mol/L solution of DIBAL-H in hexane (17.2 mL, 17.6 mmol) was addeddropwise thereto, and the mixture was stirred at −78° C. for 1 hour. Anaqueous solution of Rochelle salt was added thereto, and the mixture wasstirred, and extracted with ethyl acetate. The obtained organic layerwas washed with brine, dried over anhydrous magnesium sulfate, andconcentrated under reduced pressure. The obtained residue was dissolvedin methanol (30 mL), p-toluenesulfonic acid monohydrate (0.244 g, 1.28mmol) was added thereto, and the mixture was stirred at room temperaturefor 7 hours. The mixture was quenched with an aqueous solution of sodiumhydrogen carbonate and extracted with ethyl acetate. The obtainedorganic layer was washed with brine, dried over anhydrous magnesiumsulfate, and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (ethyl acetate-hexane) toobtain Compound 42 (2.43 g, 88%).

¹H-NMR (CDCl₃) δ: 1.42 (d, J=7.0 Hz, 3H), 3.34 (s, 3H), 3.53 (dd,J=12.0, 2.3 Hz, 1H), 3.64 (dd, J=11.5, 3.8 Hz, 1H), 3.76 (d, J=11.4 Hz,1H), 4.01 (d, J=12.0 Hz, 1H), 4.06-4.12 (m, 1H), 4.65 (d, J=5.4 Hz, 2H),5.14 (br s, 1H), 5.24 (dd, J=10.4, 1.3 Hz, 2H), 5.33 (dd, J=17.3, 1.4Hz, 2H), 5.95 (ddd, J=22.6, 10.7, 5.5 Hz, 1H).

Example 4: Method for Producing Compound 50

First Step

Compound 43 (4.00 g, 16.3 mmol) was dissolved in dichloromethane (40mL), then oxalyl dichloride (1.56 mL, 17.9 mmol) and DMF (0.013 mL,0.162 mmol) were added dropwise thereto under iced-bath, and the mixturewas warmed up to room temperature and stirred for 5 hours. The mixturewas concentrated under reduced pressure, and the obtained residue wasdissolved in dichloromethane (40 mL), then 2,2,2-trifluoroethanol (2.44g, 24.4 mmol), triethylamine (4.50 mL, 32.5 mmol) and4-(dimethylamino)pyridine (99.0 mg, 0.812 mmol) were added thereto undericed-bath, and the mixture was warmed up to room temperature and stirredfor 1 hour. The mixture was concentrated under reduced pressure, a 1mol/L aqueous solution of hydrochloric acid (100 mL) was added to theobtained residue, and the mixture was extracted with ethyl acetate (200mL). The obtained organic layer was washed with a 1 mol/L aqueoussolution of hydrochloric acid (100 mL) and brine (100 mL), dried overanhydrous magnesium sulfate to obtain Compound 44 (5.33 g, 100%).

1H-NMR (CDCl3) δ: 4.64 (q, J=8.2 Hz, 2H), 5.38 (s, 2H), 6.49 (d, J=5.6Hz, 1H), 7.30-7.38 (m, 3H), 7.43-7.49 (m, 2H), 7.75 (d, J=5.6 Hz, 1H).

Second Step

Compound 44 (5.33 g, 16.2 mmol) was dissolved in DMF (55 mL), then Bochydrazine (1.93 g, 14.6 mmol) and PPTS (12.2 g, 0.162 mmol) were addedthereto, and the mixture was stirred at 60° C. for 16 hours. Water (100mL) was added to the reaction solution, and the reaction solution wasextracted with ethyl acetate (300 mL). The obtained organic layer waswashed with water (100 mL) and brine (100 mL), and dried over anhydroussodium sulfate. The obtained organic layer was concentrated underreduced pressure, and the obtained residue was purified by silica gelcolumn chromatography (hexane-ethyl acetate) to obtain Compound 45 (1.69g, 22%).

1H-NMR (CDCl3) δ: 1.45 (s, 9H), 4.51 (q, J=8.2 Hz, 2H), 5.29 (s, 2H),6.42 (d, J=7.9 Hz, 1H), 7.28-7.37 (m, 4H), 7.39-7.43 (m, 2H), 7.68 (brs,1H).

Third Step

Compound 45 (1.59 g, 3.59 mmol) was dissolved in 4 mol/L hydrogenchloride in ethyl acetate (16 mL), and the mixture was stirred at roomtemperature for 1.5 hours. A saturated aqueous solution of sodiumhydrogen carbonate (100 mL) was added to the reaction solution, and thereaction solution was extracted with dichloromethane (200 mL) and driedover anhydrous magnesium sulfate to obtain Compound 46 (1.18 g, 96%).

1H-NMR (CDCl3) δ: 4.55 (q, J=8.3 Hz, 2H), 5.18 (s, 2H), 5.29 (s, 2H),6.37 (d, J=7.8 Hz, 1H), 7.30-7.42 (m, 6H).

Fourth Step

Compound 46 (1.18 g, 3.45 mmol) and Compound 42 (890 mg, 4.14 mmol) weredissolved in acetonitrile (24 mL), tin tetrachloride (0.607 mL, 5.17mmol) was added dropwise thereto at −30° C., and the mixture was stirredat −30° C. for 1 hour. A saturated aqueous solution of sodium hydrogencarbonate (200 mL) and dichloromethane (200 mL) were added to thereaction solution, and unwanted matter was filtered off. The obtainedorganic layer was washed with brine (100 mL) and dried over anhydroussodium sulfate. The obtained organic layer was concentrated underreduced pressure, and the obtained residue was purified by silica gelcolumn chromatography (chloroform-methanol) to obtain Compound 47 (1.22g, 67%). LC/MS (ESI): m/z=525.9 [M+H]⁺, RT=2.02 min, method (1)

Fifth Step

Compound 47 (1.15 g, 2.19 mmol) was dissolved in THF (23 mL), thenmorpholine (0.953 mL, 10.9 mmol) and tetrakistriphenylphosphinepalladium(126 mg, 0.109 mmol) were added thereto under nitrogen atmosphere, andthe mixture was stirred at room temperature for 7.5 hours. The mixturewas concentrated under reduced pressure, and the obtained residue waspurified by silica gel column chromatography (chloroform-methanol) toobtain Compound 48 (890 mg, quant.).

LC/MS (ESI): m/z=342.1 [M+H]⁺, RT=1.00, 1.09 min, method (1)

Sixth Step

Compound 48 (820 mg, 2.40 mmol) was dissolved in dichloromethane (16.5mL), then Boc₂O (0.837 mL, 3.60 mmol), triethylamine (0.499 mL, 3.60mmol) and 4-(dimethylamino)pyridine (44.0 mg, 0.360 mmol) were addedthereto, and the mixture was stirred at room temperature for 3.5 hours.A 1 mol/L aqueous solution of hydrochloric acid (50 mL) was added to thereaction solution, and the mixture was extracted with ethyl acetate (125mL). The obtained organic layer was washed with a 1 mol/L aqueoussolution of hydrochloric acid (50 mL) and brine (50 mL), and dried overanhydrous sodium sulfate. The obtained organic layer was concentratedunder reduced pressure, and the obtained residue was purified by silicagel column chromatography (chloroform-methanol) to obtain Compound 49(593 mg, 56%) and Compound 50 (170 mg, 16%).

LC/MS (ESI): m/z=441.9 [M+H]⁺, RT=1.67 min, method (1)

Seventh Step

Compound 49 (547 mg, 1.24 mmol) was dissolved in acetic acid (5.5 mL),and the mixture was stirred at 80° C. for 5 hours. The mixture wasconcentrated under reduced pressure, and the obtained residue waspurified by silica gel column chromatography (chloroform-methanol) toobtain Compound 50 (454 mg, quant.).

1H-NMR (CDCl3) δ: 1.46 (d, J=6.4 Hz, 3H), 3.45 (dd, J=10.5, 10.5 Hz,1H), 3.55 (dd, J=11.7, 4.3 Hz, 1H), 3.92 (dd, J=11.7, 3.6 Hz, 1H),3.95-4.01 (m, 2H), 4.76 (dq, J=13.9, 4.3 Hz, 1H), 5.19 (d, J=10.2 Hz,1H), 5.22 (d, J=10.2 Hz, 1H), 5.36 (d, J=12.9 Hz, 1H), 6.28 (d, J=7.8Hz, 1H), 7.25 (d, J=7.8 Hz, 1H), 7.28-7.36 (m, 3H), 7.56-7.61 (m, 2H).

Example 5: Method for Producing Compound 55

First Step

To a suspension of Compound 51 (19.2 g, 77.8 mmol) and potassiumcarbonate (16.13 g, 117 mmol) in acetone (190 mL) was added thiophenol(8.01 mL, 78 mmol), and the mixture was stirred at 40° C. for 1 hour.The reaction solution was cooled to 25° C. and ethyl acetate and waterwere added thereto. The mixture was extracted with ethyl acetate, andthe obtained organic layer was washed twice with water and concentratedunder reduced pressure to obtain Compound 52.

¹H-NMR (CDCl₃) δ: 3.93 (3H, s), 4.93 (2H, s), 7.03-7.07 (1H, m),7.18-7.35 (6H, m), 7.93-8.06 (1H, m).

Second Step

To a solution of Compound 52 (21.5 g, 77.8 mmol) in methanol (60 mL) andTHF (40 mL) was added dropwise 2 mol/L sodium hydroxide (97.0 mL, 195mmol) under ice bath, and the reaction solution was left to stand stillall night. The reaction solution was concentrated under reduced pressureand water was added thereto. The obtained aqueous layer was washed twicewith hexane. The aqueous layer was made acidic with 6 mol/L hydrochloricacid and extracted twice with ethyl acetate. The obtained organic layerwas dried with sodium sulfate and concentrated under reduced pressure.The obtained residue was crystallized with ethyl acetate/hexane toobtain Compound 53 as crystals (7.7 g).

¹H-NMR (CDCl₃) δ: 4.40 (2H, s), 6.81-6.84 (1H, m), 7.07-7.32 (6H, m),7.86-7.90 (1H, m).

Third Step

To polyphosphoric acid (200 g, 29.4 mmol) was added Compound 53 (7.70 g,29.4 mmol) at 60° C., and the reaction mixture was warmed up to 140° C.and stirred for 1 hour. The reaction solution was cooled to 40° C., andwater was added thereto under iced-bath. The slurry was filtered, andthe filtrate was washed with water. Ethyl acetate was added to thefiltrate, and the obtained organic layer was washed with water andbrine, dried over sodium sulfate, and concentrated under reducedpressure. The obtained residue was crystallized with ethylacetate/hexane to obtain Compound 54 as crystals (3.6 g).

¹H-NMR (CDCl₃) δ: 4.03 (2H, s), 6.92-7.06 (2H, m), 7.26-7.40 (4H, m),7.67 (1H, dd, J=5.5 Hz, J=8.0 Hz), 8.25 (1H, d, J=8.0 Hz).

Fourth Step

To a solution of Compound 54 (3.60 g, 14.7 mmol) in methanol (14 mL) andTHF (28 mL) was added sodium hydrogen borate (613 mg, 16.2 mmol) underice-bath. The reaction solution was stirred at room temperature for 30minutes. Water was added thereto, and the reaction solution was left tostand still all night. The reaction solution was concentrated underreduced pressure, and ethyl acetate and water were added to theconcentrate. The mixture was extracted. The obtained organic layer waswashed with brine, dried over sodium sulfate, and concentrated underreduced pressure. The obtained residue was purified by silica gel columnchromatography (methylene chloride) to obtain Compound 55 (1.7 g) ascrystals.

¹H-NMR (CDCl₃) δ: 4.15 (1H, d, J=14.0 Hz), 4.57 (1H, d, J=14.0 Hz), 6.09(1H, s), 6.91-6.93 (2H, m), 7.10-7.17 (3H, m), 7.39 (1H, dd, J=5.5 Hz,J=8.0 Hz), 7.48 (1H, d, J=8.0 Hz).

Example 6: Method for Producing Compound III-30

First Step

Compound 50 (154.0 mg, 0.451 mmol) and Compound 55 (117 mg, 0.474 mmol)were dissolved in a solution of T3P in ethyl acetate (1.5 mL), and themixture was stirred in a sealed tube at 100° C. for 4 hours. Thereaction was stopped by adding water, the reaction solution wasextracted with ethyl acetate, and the obtained organic layer was washedwith brine, dried over anhydrous magnesium sulfate, and concentratedunder reduced pressure. The obtained residue was recrystallized withchloroform-hexane to obtain Compound 56 (70.2 mg, 27%).

LC/MS (ESI): m/z=570 [M+H]⁺, RT=2.18 min, method (1)

Second Step

Compound 56 (70.2 mg, 0.123 mmol) was dissolved in DMA (1 mL), lithiumchloride (52.2 g, 1.23 mmol) was added thereto, and the mixture wasstirred at 100° C. for 4 hours. The mixture was cooled to roomtemperature, and a 1 mol/L aqueous solution of hydrochloric acid wasadded thereto to stop the reaction. The reaction solution was extractedwith ethyl acetate, the obtained organic layer was washed with 1 mol/Lhydrochloric acid and brine, dried over anhydrous sodium sulfate, andconcentrated under reduced pressure. The obtained solids were washedwith ethyl acetate to obtain Compound III-30 (28.6 mg, 48%).

1H-NMR (CDCl3) δ: 1.78 (d, J=7.2 Hz, 3H), 3.26-3.32 (m, 1H), 3.44-3.60(m, 3H), 3.72 (dd, J=11.7, 2.6 Hz, 1H), 3.94 (dd, J=11.2, 2.9 Hz, 1H),4.42 (dd, J=9.9, 2.8 Hz, 1H), 5.29 (s, 1H), 5.54 (d, J=13.6 Hz, 1H),5.76 (d, J=7.8 Hz, 1H), 6.71 (d, J=7.7 Hz, 1H), 6.81-6.86 (m, 1H),6.96-7.04 (m, 2H), 7.07-7.11 (m, 3H), 7.23-7.25 (m, 1H).

LC/MS (ESI): m/z=480 [M+H]⁺, RT=1.87 min, method (1)

Example 7: Method for Producing Compound III-54

First Step

Compound 57 (60.6 g, 0.162 mol) was dissolved in THF (122 ml), thenCompound 58 (Journal of Organic Chemistry, 80(20), 9868-9880; 2015) (50g, 0.34 mol) and DBU (2.6 g, 17 mmol) were added thereto, and themixture was stirred at 60° C. for 48 hours. The reaction solution wasconcentrated, and purified by silica gel column chromatography(dichloromethane-methanol) to obtain Compound 59 (73.2 g, 93.3%).

Second Step

Compound 59 (73.2 g, 0.15 mol) was dissolved in a mixed solvent ofacetonitrile (580 ml) and water (145 ml), methanesulfonic acid (43.1 g,0.45 mol) was added thereto, and the mixture was stirred at 60° C. for20 hours. The mixture was concentrated under reduced pressure until theresidue was about 300 ml. Ethyl acetate (250 ml) and an aqueous solutionof sodium carbonate (500 ml) were added thereto, the pH of the mixturewas regulated to 8, and the mixture was stirred for 30 minutes.Precipitates were filtered, and washed with a mixed solvent of ethylacetate-hexane and water, and dried to obtain Compound 60 (33 g, 68%).

1H NMR (400 MHz, d-DMSO) δ:7.70 (d, J=7.6 Hz, 1H), 7.55 (d, J=7.1 Hz,2H), 7.40-7.22 (m, 4H), 6.21 (d, J=7.6 Hz, 1H), 5.05 (s, 2H), 4.59 (d,J=3.6 Hz, 1H), 4.39-4.27 (m, 1H), 2.80-2.65 (m, 1H), 1.99-1.87 (m, 1H),1.78 (s, 2H), 1.51-1.15 (m, 3H).

Third Step

The optical resolution of Compound 60 (4.0 g, 12.3 mmol) by Waters SFC30System (Daicel CHIRALPAK IB, liquefied carbon dioxide-methanol) gaveCompound 61 (1.79 g, 45%).

Analysis Condition

<Waters SFC30 System>

Column: CHIRALPAK IB/SFC (5 μm, i.d. 250×4.6 mm) (DAICEL)

Flow rate: 8.0 mL/mini UV detection wavelength: 254 nm

Back pressure: 100 bar

Mobile phase: [A]: liquefied carbon dioxide, [B]: methanol

Gradient: 5% solvent. [B] was kept, for 1 minute, a linear gradient, of5% to 40% solvent [B] was carried out in 6 minutes, 40% solvent [B] waskept for 2 minutes, and 5% solvent [B] was kept for 1 minute.

Elution time: 7.9 minutes

Fourth Step

Compound 61 (1.76 g, 5.41 mmol) and8-fluoro-6,11-dihydrodibenzo[b,e]thiepin-11-ol (1.99 g, 8.11 mmol) weredissolved in T3P (50%, ethyl acetate solution, 16 ml), and the mixturewas stirred in a sealed tube at 100° C. for 3 hours. Ethyl acetate andwater were added thereto, the mixture was neutralized with a saturatedaqueous solution of sodium hydrogen carbonate, and the aqueous layer wasextracted with ethyl acetate. The obtained organic layer was washed withbrine, dried over anhydrous sodium sulfate, and concentrated underreduced pressure. The obtained residue was dissolved in THF (15 ml),then potassium carbonate (1.49 g, 10.8 mmol) and benzyl bromide (0.321ml) were added thereto, and the mixture was heated to reflux for 6hours. Further, 1-methylpiperazine (0.30 ml) was added thereto, and themixture was heated to reflux for 1 hour. The reaction solution wasextracted with ethyl acetate. The obtained organic layer was washed with2 mol/L hydrochloric acid and brine, dried over anhydrous magnesiumsulfate, and concentrated under reduced pressure. The obtained residuewas purified by silica gel column chromatography (chloroform-acetone) toobtain Compound 62 (1.15 g, 38.4%).

LC/MS (ESI): m/z=554 [M+H]⁺, RT=2.24 min. method (1)

Fifth Step

Compound 62 (1.0 g, 1.81 mol) and lithium chloride (383 mg, 9.03 mmol)were dissolved in DMA, and the mixture was stirred at 100° C. for 3hours. The reaction solution was cooled to room temperature, acetone (5ml) was added thereto, a 1 mol/L aqueous solution of hydrochloric acid(40 ml) was added dropwise thereto, and the mixture was stirred at roomtemperature for 15 minutes. The produced white solids were filtered,washed with a 50% aqueous solution of acetone, and dried to obtainCompound III-54 (760 mg, 91%).

¹H-NMR (CDCl₃) δ: 1.47-2.05 (m, 6H), 2.50-2.58 (m, 1H), 3.51 (d, J=12.0Hz, 1H), 4.26-4.31 (m, 1H), 4.68-4.74 (m, 1H), 5.22 (s, 1H), 5.62 (d,J=13.6 Hz, 1H), 5.77 (d, J=7.6 Hz, 1H), 6.68 (d, J=7.6 Hz, 1H),6.80-6.82 (m, 1H), 6.88-7.02 (m, 1H), 7.03-7.15 (m, 5H) LC/MS (ESI):m/z=464.2 [M+H]⁺, RT=1.92 min, method (1)

Example 8: Method for Producing Compound III-20

First Step

Compound i1 (500 mg, 1.53 mmol) and10-chloro-6,11-dihydrodibenzo[b,e]thiepin-11-ol (602 mg, 2.29 mmol) weredissolved in T3P (50%, ethyl acetate solution, 5 ml), and the mixturewas stirred in a sealed tube at 105° C. for 1.5 hours. The reactionsolution was extracted with ethyl acetate, the obtained organic layerwas washed with water and brine, dried over anhydrous magnesium sulfate,and concentrated under reduced pressure. The obtained residue waspurified by silica gel column chromatography (chloroform-ethylacetate-methanol) to obtain Compound 63 (210 mg, 24%).

1H-NMR (CDCl3) δ: 2.96 (t, J=12.0 Hz, 1H), 3.30-3.43 (m, 2H), 3.53 (d,J=13.2 Hz, 1H), 3.73 (d, J=11.6 Hz, 1H), 3.86 (d, J=10.8 Hz, 1H), 4.42(d, J=7.2 Hz, 1H), 4.71 (d, J=13.6 Hz, 1H), 5.42 (d, J=10.8 Hz, 1H),5.57-5.65 (m, 2H), 5.79 (d, J=7.6 Hz, 1H), 6.18 (s, 1H), 6.47 (d, J=7.2Hz, 1H), 6.67 (t, J=6.8 Hz, 1H), 7.00-7.14 (m, 3H), 7.22-7.40 (m, 6H),7.64 (d, J=7.2 Hz, 2H).

Second Step

Compound 63 (210 mg, 0.368 mmol) was dissolved in DMA (3 ml), lithiumchloride (78 mg, 1.84 mmol) was added thereto, and the mixture wasstirred at 100° C. for 4 hours. The reaction solution was extracted withethyl acetate, the obtained organic layer was washed with 1 mol/Lhydrochloric acid and brine, dried over anhydrous sodium sulfate, andconcentrated under reduced pressure. The obtained residue wasrecrystallized with dichloromethane-diethyl ether to obtain CompoundIII-20 (124 mg, 70%).

1H-NMR (CDCl3) δ: 3.09 (t, J=12.8 Hz, 1H), 3.48 (t, J=11.6 Hz, 1H),3.55-3.62 (m, 2H), 3.81 (d, J=11.6 Hz, 1H), 3.93 (d, J=10.8 Hz, 1H),4.53 (d, J=9.6 Hz, 1H), 4.69 (d, J=13.2 Hz, 1H), 5.68 (d, J=12.8 Hz,1H), 5.76 (d, J=6.8 Hz, 1H), 6.26 (s, 1H), 6.80-6.88 (m, 2H), 7.05-7.15(m, 3H), 7.24-7.28 (m, 1H), 7.34 (t, J=7.6 Hz, 1H), 7.39 (d, J=8.0 Hz,1H).

Example 9: Method for Producing Compound III-33

First Step

Compound 64 (3.54 g, 14.9 mmol) was dissolved in dichloromethane (70ml), and DIBAL-H (1 mol/L, 16.4 ml, 16.4 mmol) was added dropwisethereto at −78° C. The mixture was stirred at −78° C. for 1 hour, thereaction was quenched with methanol, and the temperature of the mixturewas increased to room temperature. The reaction mixture was extractedwith dichloromethane, the obtained organic layer was washed with 2 mol/Lhydrochloric acid and brine, dried over anhydrous magnesium sulfate, andconcentrated under reduced pressure. The obtained residue was dissolvedin methanol (35 ml), ammonium chloride (80 mg, 1.49 mmol) was addedthereto, and the mixture was heated to reflux for 1 hour. The reactionmixture was extracted with diethyl ether, and the obtained organic layerwas washed with a saturated aqueous solution of sodium hydrogencarbonate and brine, dried over anhydrous magnesium sulfate, andconcentrated under reduced pressure to obtain Compound 65 (2.73 g, 76%).

¹H-NMR (CDCl3) δ: 0.96 (s, 6H), 1.53 (d, J=5.2 Hz, 2H), 1.85-1.90 (m,2H), 3.31 (s, 6H), 3.38-3.43 (m, 2H), 4.44 (t, J=5.2 Hz, 1H)

Second Step

Compound 65 (2.7 g, 11.3 mmol) was dissolved in acetone (50 ml), thenpotassium carbonate (5.46 g), isoindoline-1,3-dione (2.49 g, 16.9 mmol)and tetrabutylammonium bromide (1.09 g, 3.39 mmol) were added thereto,and the mixture was healed to reflux for 6 hours. The reaction solutionwas filtered and concentrated under reduced pressure. The obtainedresidue was purified by silica gel column chromatography (hexane-ethylacetate) to obtain Compound 66 (1.1 g, 32%).

¹H-NMR (CDCl₃) δ: 1.04 (s, 6H), 1.58-1.65 (m, 4H), 3.32 (s, 6H),3.65-3.75 (m, 2H), 4.52 (t, J=5.2 Hz, 1H), 7.65-7.75 (m, 2H), 7.80-7.90(m, 2H)

Third Step

Compound 66 (1.14 g, 3.73 mmol) was dissolved in ethanol-water (VI, 4ml), hydrazine monohydrate (374 mg, 7.47 mmol) was added thereto, andthe mixture was stirred at 60° C. for 5 hours. The reaction solution wasextracted with dichloromethane, and the obtained organic layer waswashed with a 1 mol/L aqueous solution of sodium hydroxide and brine,dried over anhydrous magnesium sulfate, and concentrated under reducedpressure to obtain Compound 67 (750 mg, 100%).

¹H-NMR (CDCl₃) δ: 0.93 (s, 6H), 1.38-1.45 (m, 2H), 1.52 (d, J=5.2 Hz,2H), 2.65-2.75 (m, 2H), 3.30 (s, 6H), 4.46 (t, J=5.2 Hz, 1H), 5.30 (s,2H)

Fourth Step

Compound 57 (300 mg, 0.80 mmol) was dissolved in THF (0.6 ml), thenCompound 67 (351 mg, 2.00 mmol) and DBU (12 μl, 0.08 mmol) were addedthereto, and the mixture was stirred at 60° C. for 18 hours. Thereaction solution was concentrated under reduced pressure, and theobtained residue was purified by silica gel column chromatography(chloroform-methanol) to obtain Compound 68 (440 mg, 80%).

¹H-NMR (CDCl₃) δ: 0.90 (s, 6H), 1.20-1.32 (m, 2H), 1.44 (s, 9H), 1.47(d, J=5.2 Hz, 2H), 3.11-3.25 (m, 2H), 3.26 (s, 6H), 4.41 (t, J=5.21 Hz,1H), 5.28 (s, 2H), 6.38 (d, J=8.0, 1H), 6.87 (br, 1H), 7.29-7.40 (m,6H), 8.49 (br, 1H)

Fifth Step

Compound 68 (210 mg, 0.41 mmol) was dissolved in a mixed solvent ofacetonitrile (1.8 ml) and water (310 μl), methanesulfonic acid (79 μl,1.22 mmol) was added thereto, and the mixture was stirred at 60° C. for6 hours. The mixture was cooled to room temperature, the pH of themixture was regulated to 7 with a 2 mol/L aqueous solution of sodiumhydroxide, and the reaction solution was extracted with dichloromethane.The obtained organic layer was washed with brine, dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure. The obtainedresidue was purified by silica gel column chromatography(chloroform-methanol) to obtain Compound 69 (55 mg, 49%).

¹H-NMR (CDCl₃) δ: 0.95 (s, 3H), 0.98 (s, 3H), 1.40-1.45 (m, 2H),1.53-1.65 (m, 2H), 2.80-2.89 (m, 1H), 4.05-4.15 (m, 1H), 4.40-4.48 (m,1H), 5.14-5.31 (m, 2H), 5.48 (d, J=12.4 Hz, 1H), 6.32 (d, J=7.6 Hz, 1H),7.25-7.40 (m, 4H), 7.59 (d, J=6.8 Hz, 2H)

Sixth Step

The optical resolution of Compound 69 (6.69 g, 18.9 mmol) by WatersSFC30 System (Daicel CHIRALPAK IB, liquefied carbon dioxide-methanol)gave Compound 70 (3.40 g, 50%).

Analysis Condition

<Waters SFC30 System>

Column: CHIRALPAK IB/SFC (5 μm, i.d. 250×4.6 mm) (DAICEL)

Flow rate: 8.0 mL/min; UV detection wavelength: 254 nm

Back pressure: 100 bar

Mobile phase: [A]: liquefied carbon dioxide, [B]: methanol

Gradient: 5% solvent [B] was kept for 1 minute, a linear gradient of 5%to 40% solvent [B] was carried out in 6 minutes, 40% solvent [b] waskept for 2 minutes, and 5% solvent [B] was kept for 1 minute.

Elution time: 7.67 minutes

Seventh Step

Compound 70 (2.0 g, 5.66 mmol) and7,8-difluoro-6,11-dihydrodibenzo[b,e]thiepin-11-ol (1.94 g, 7.36 mmol)were dissolved in T3P (50% ethyl acetate, 20 ml), and the mixture wasstirred in a sealed tube at 105° C. for 2.5 hours. The reaction solutionwas extracted with ethyl acetate, and the obtained organic layer waswashed with water and brine, dried over anhydrous magnesium sulfate, andconcentrated under reduced pressure. The obtained residue was purifiedby silica gel column chromatography (chloroform-acetone) to obtain adiastereomeric mixture (2.84 g). This was recrystallized with ethylacetate to obtain Compound 71 (1.26 g, 37%).

¹H-NMR (CDCl3) δ: 0.80 (s, 3H), 0.91 (s, 3H), 1.20-1.45 (m, 4H),2.49-2.58 (m, 1H), 4.02 (d, J=13.6 Hz, 1H), 4.25-4.33 (m, 1H), 4.55-4.65(m, 1H), 5.19 (s, 1H), 5.24-5.30 (m, 1H), 5.47-5.59 (m, 2H), 5.80 (d,J=7.6 Hz, 1H), 6.42 (d, J=8.0 Hz, 1H), 6.65-6.71 (m, 1H), 6.95-7.12 (m,5H), 7.27-7.39 (m, 3H), 7.61 (d, J=6.8 Hz, 2H)

Eighth Step

Compound 71 (980 mg, 1.63 mmol) was dissolved in DMA (8 ml), lithiumchloride (346 mg, 8.17 mmol) was added thereto, and the mixture wasstirred at 100° C. for 3 hours. The reaction solution was extracted withethyl acetate, the obtained organic layer was washed with 2 mol/Lhydrochloric acid and brine, dried over anhydrous sodium sulfate, andconcentrated under reduced pressure. The obtained residue wascrystallized with ethyl acetate to obtain Compound III-33 (780 mg, 94%).

¹H-NMR (CDCl₃) δ: 0.85 (s, 3H), 0.97 (s, 3H), 1.34-2.00 (m, 4H),2.62-2.66 (m, 1H), 4.05 (d, J=13.6 Hz, 1H), 4.40-4.48 (m, 1H), 4.56-4.63(m, 1H), 5.24 (s, 1H), 5.30-5.35 (m, 1H), 5.80 (d, J=7.6 Hz, 1H), 6.68(d, J=7.6 Hz, 1H), 6.78-6.90 (m, 1H), 6.95-7.15 (m, 4H), 7.16-7.22 (m,1H)

LC/MS (ESI): m/z=510.2 [M+H]⁺, RT=2.25 min, method (1)

The following example compounds were synthesized from commerciallyavailable compounds or intermediates suitably synthesized fromcommercially available compounds according to the above examples.

TABLE 1 No. Structure H-NMR or LC/MS III-3

1H-NMR (CDCl3) δ: 2.99 (t, J = 12.4 Hz, 1H), 3.43- 3.61 (m, 3H), 3.81(d, J = 12.0 Hz, 1H), 3.96 (d, J = 11.0 Hz, 1H), 4.59 (d, J = 9.8 Hz,1H), 4.66 (d, J = 13.2 Hz, 1H), 5.26 (s, 1H), 5.54 (d, J = 13.4 Hz, 1H),5.75 (d, J = 8.2 Hz, 1H), 6.69 (d, J = 7.7 Hz, 1H), 6.84 (t, J = 7.0 Hz,1H), 6.98-7.05 (m, 2H), 7.07- 7.12 (m, 3H), 7.22 (t, J = 7.0 Hz, 1H).III-9

1H-NMR(CDCl3) δ: 2.37 (d, J = 13.2 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H),2.79-2.87 (m, 1H), 2.90-3.03 (m, 2H), 4.08 (d, J = 13.6 Hz, 1H), 4.64(d, J = 10.8 Hz, 1H), 5.05 (d, J = 12.0 Hz, 1H), 5.19 (s, 1H), 5.25-5.32(m, 1H), 5.78 (d, J = 7.6 Hz, 1H), 6.66 (d, J = 7.6 Hz, 1H), 6.84 (t, J= 7.6 Hz, 1H), 6.90-7.20 (m, 5H). III-10

1H-NMR(CDCl3) δ: 3.06 (t, J = 11.6 Hz, 1H), 3.47 (t, J = 11.2 Hz, 1H),3.50-3.63 (m, 2H), 3.80 (d, J = 11.6 Hz, 1H), 3.94 (d, J = 11.2 Hz, 1H),4.58 (d, J = 9.6 Hz, 1H), 4.69 (d, J = 13.6 Hz, 1H), 5.57 (d, J = 13.6Hz, 1H), 5.75 (d, J = 7.6 Hz, 1H), 5.90 (s, 1H), 6.78 (d, J = 7.6 Hz,1H), 6.85 (t, J = 7.6 Hz, 1H), 7.04-7.17 (m, 5H), 7.35-7.42 (m, 1H).III-11

1H-NMR(CDCl3) δ: 3.04 (t, J = 12.0 Hz, 1H), 3.47 (t, J = 11.6 Hz, 1H),3.59 (t, J = 11.2 Hz, 1H), 3.82 (d, J = 12.0 Hz, 1H), 3.97 (d, J = 10.8Hz, 1H), 4.03 (d, J = 14.0 Hz, 1H), 4.56 (d, J = 11.6 Hz, 1H), 4.68 (d,J = 13.6 Hz, 1H), 5.17 (d, J = 14.0 Hz, 1H), 5.24 (s, 1H), 5.75 (d, J =8.0 Hz, 1H), 6.69 (d, J = 7.6 Hz, 1H), 6.80-6.88 (m, 2H), 6.98 (t, J =8.8 Hz, 1H), 7.04-7.16 (m, 3H). III-12

1H-NMR(CDCl3) δ: 3.04 (t, J = 12.8 Hz, 1H), 3.40- 3.62 (m, 3H), 3.82 (d,J = 12.0 Hz, 1H), 3.96 (d, J = 11.2 Hz, 1H), 4.58 (d, J = 9.6 Hz, 1H),4.68 (d, J = 13.6 Hz, 1H), 5.19 (s, 1H), 5.49 (d, J = 13.6 Hz, 1H), 5.74(d, J = 7.6 Hz, 1H), 6.68 (d, J = 7.2 Hz, 1H), 6.85 (t, J = 7.6 Hz, 1H),7.03 (d, J = 7.6 Hz, 1H), 7.06-7.16 (m, 3H), 7.21 (t, J = 8.8 Hz, 1H).III-13

1H-NMR(CDCl3) δ: 3.04 (t, J = 12.0 Hz, 1H), 3.47 (t, J = 12.0 Hz, 1H),3.58 (t, J = 10.8 Hz, 1H), 3.69 (d, J = 13.6 Hz, 1H), 3.81 (d, J = 12.0Hz, 1H), 3.94 (d, J = 11.2 Hz, 1H), 4.57 (d, J = 13.6 Hz, 1H), 4.69 (d,J = 14.0 Hz, 1H), 5.59 (d, J = 13.6 Hz, 1H), 5.79 (d, J = 7.6 Hz, 1H),5.96 (s, 1H), 6.63 (d, J = 7.6 Hz, 1H), 6.81-6.88 (m, 1H), 6.96 (t, J =9.6 Hz, 1H), 7.04-7.13 (m, 2H), 7.17 (d, J = 7.6 Hz, 1H), 7.38- 7.45 (m,1H).

TABLE 2 III-14

1H-NMR (CDCl3) δ: 3.00-3.07 (m, 1H), 3.47 (td, J = 12.0, 2.6 Hz, 1H),3.57-3.62 (m, 2H), 3.82 (dd, J = 11.9, 3.3 Hz, 1H), 3.97 (dd, J = 11.1,2.9 Hz, 1H), 4.60 (dd, J = 10.0, 3.0 Hz, 1H), 4.68 (dd, J = 13.6, 2.0Hz, 1H), 5.20 (s, 1H), 5.47 (d, J = 13.4 Hz, 1H), 5.76 (d, J = 7.8 Hz,1H), 6.70 (d, J = 7.8 Hz, 1H), 6.82-6.86 (m, 1H), 6.98 (dd, J = 8.7, 2.5Hz, 1H), 7.07- 7.16 (m, 4H), 7.35 (dd, J = 8.3, 5.5 Hz, 1H). III-21

1H-NMR(CDCl3) δ:1.85-1.98 (m, 1H), 2.10-2.23 (m, 2H), 2.31-2.43 (m, 1H),2.69 (t, J = 10.8 Hz, 1H), 4.09 (d, J = 13.2 Hz, 1H), 4.51 (d, J = 12.4Hz, 1H), 4.77 (d, J = 13.6 Hz, 1H), 5.20-5.30 (m, 1H), 5.78 (d, J = 7.2Hz, 1H), 5.77 (d, J = 7.6 Hz, 1H), 6.68 (d, J = 7.2 Hz, 1H), 6.81-6.88(m, 1H), 6.96-7.02 (m, 1H), 7.05-7.17 (m, 4H). III-22

1H-NMR(CDCl3) δ: 1.22 (d, J = 7.2 Hz, 3H), 3.49-3.58 (m, 4H), 3.95 (dd,J = 10.8, 2.8 Hz, 1H), 4.08 (d, J = 13.8 Hz, 1H), 4.74 (dd, J = 10.0,2.8 Hz, 1H), 4.99- 5.05 (m, 1H), 5.22 (s, 1H), 5.30 (dd, J = 13.8, 2.3Hz, 1H), 5.75 (d, J = 7.8 Hz, 1H), 6.69 (d, J = 7.7 Hz, 1H), 6.84 (t, J= 7.0 Hz, 1H), 6.97-7.02 (m, 2H), 7.08-7.14 (m, 3H). III-23

1H-NMR(CDCl3) δ: 1.29-1.87 (m, 8H), 2.67 (td, J = 13.5, 2.6 Hz, 1H),3.54-3.66 (m, 5H), 4.08 (d, J = 13.7 Hz, 1H), 4.47 (dd, J = 12.0, 2.3Hz, 1H), 4.61 (dd, J = 13.8, 3.1 Hz, 1H), 5.24-5.33 (m, 2H), 5.79 (d, J= 7.8 Hz, 1H), 6.68 (d, J = 7.5 Hz, 1H), 6.83-6.87 (m, 1H), 6.98-7.15(m, 5H). III-26

1H-NMR (CDCl3) δ: 1.82-2.17 (5H, m), 2.59-2.76 (1H, m), 2.84 (1H, t, J =11.5 Hz) 4.09 (1H, d, J = 13.8 Hz), 4.63-4.69 (2H, m), 5.22 (1H, s),5.27 (1H, dd, J = 13.9, 2.4 Hz), 5.79 (1H, d, J = 7.7 Hz), 6.68 (1H, d,J = 7.7 Hz), 6.83-6.87 (1H, m), 7.15-6.96 (5H, m). III-28

1H-NMR (CDCl3) δ: 1.79 (d, J = 7.2 Hz, 3H), 3.33-3.40 (m, 1H), 3.46-3.75(m, 5H), 3.94 (dd, J = 11.0, 2.9 Hz, 1H), 4.43 (dd, J = 9.7, 2.7 Hz,1H), 5.58 (d, J = 13.6 Hz, 1H), 5.81 (d, J = 7.7 Hz, 1H), 6.00 (s, 1H),6.65 (d, J = 7.7 Hz, 1H), 6.82-6.88 (m, 1H), 6.94-7.01 (m, 2H), 7.11 (t,J = 9.2 Hz, 1H), 7.17 (d, J = 7.5 Hz, 1H), 7.39-7.44 (m, 1H) III-29

1H-NMR (CDCl3) δ: 1.62-1.69 (m, 1H), 1.90 (t, J = 12.4 Hz, 1H), 2.13 (d,J = 13.7 Hz, 1H), 2.38-2.46 (m, 2H), 4.09-4.20 (m, 3H), 4.32 (d, J = 6.3Hz, 1H), 4.37-4.41 (m, 2H), 4.71 (dd, J = 13.7, 3.4 Hz, 1H), 5.23 (s,1H), 5.36 (dd, J = 13.7, 2.6 Hz, 1H), 5.79 (d, J = 7.8 Hz, 1H), 6.68 (d,J = 7.8 Hz, 1H), 6.82-6.87 (m, 1H), 6.94-6.99 (m, 1H), 7.05-7.15 (m,4H).

TABLE 3 III-31

LC/MS (ESI): m/z = 480 [M + H]⁺. RT = 1.81 min. method (1) III-34

1H-NMR (CDCl3) δ: 1.86-2.18 (4H, m), 2.30- 2.46 (1H, m), 2.90 (1H, dd, J= 30.0, 13.9 Hz), 4.07 (1H, d, J = 13.7 Hz), 4.41-4.48 (1H, m),4.99-5.06 (1H, m), 5.20 (1H, s), 5.30 (1H, dd, J = 13.7, 2.4 Hz), 5.78(1H, d, J = 7.8 Hz), 6.68 (1H, d, J = 7.8 Hz), 6.83-6.87 (1H, m), 7.00(1H, dd, J = 8.3, 4.1 Hz), 7.06-7.17 (4H, m). III-35

1H-NMR(CDCl3) δ: 0.89 (s, 3H), 0.95 (s, 3H), 1.25-2.20 (m, 4H), 2.39 (d,J = 12.4 Hz, 1H), 4.05 (d, J = 12.4 Hz, 1H), 4.20-4.28 (m, 1H),4.39-4.44 (m, 1H), 5.20 (m, 1H), 5.33-5.38 (m, 1H), 5.78 (d, J = 7.6 Hz,1H), 6.68 (d, J = 7.6 Hz, 1H), 6.80-6.83 (m, 1H), 6.88-7.18 (m, 5H)III-36

1H-NMR(CDCl3) δ: 0.18-0.25 (m, 1H), 0.26- 0.35 (m, 1H), 0.36-0.50 (m,2H), 0.76-0.83 (m, 1H), 0.98-1.40 (m, 1H), 1.60-2.24 (m, 4H), 2.60-2.70(m, 1H), 4.04 (d, J = 13.6 Hz, 1H), 4.32-4.48 (m, 1H), 4.69-4.75 (m,1H), 5.26 (s, 1H), 5.77 (d, J = 8.0 Hz, 1H), 6.69 (d, J = 8.0 Hz, 1H),6.80-6.90 (m, 1H), 7.00-7.18 (m, 5H)

TABLE 4 III-37

1H-NMR (CDCl3) δ: 3.26 (dd, J = 14.6, 5.7 Hz, 1H), 3.85-4.11 (m, 4H),4.68 (dd, J = 10.4, 3.6 Hz, 1H), 5.07 (d, J = 14.7 Hz, 1H), 5.22-5.27(m, 2H), 5.74 (d, J = 7.7 Hz, 1H), 6.69 (d, J = 7.5 Hz, 1H), 6.85 (t, J= 6.9 Hz, 1H), 6.97-7.15 (m, 5H). III-38

1H-NMR (CDCl3) δ: 1.49-1.79 (m, 2H), 1.91 (d, J = 11.9 Hz, 1H),2.08-2.13 (m, 1H), 2.47-2.62 (m, 2H), 4.07-4.10 (m, 1H), 4.35 (dd, J =11.9. 2.3 Hz, 1H), 4.84 (dd, J = 13.4, 4.0 Hz, 1H), 5.25 (s, 1H), 5.31(dd, J = 13.9, 2.4 Hz, 1H), 5.79 (d, J = 7.7 Hz, 1H), 6.69 (d, J = 7.9Hz, 1H), 6.83-6.87 (m, 1H), 6.97- 7.00 (m, 1H), 7.06-7.15 (m, 4H).III-39

1H-NMR(CDCl3) δ: 1.31-1.44 (m, 1H), 1.58 (q, J = 11.6 Hz, 1H), 2.05 (d,J = 10.8 Hz, 1H), 2.26 (d, J = 11.6 Hz, 1H), 2.47 (t, J = 11.2 Hz, 1H),3.31 (s, 3H), 3.40-3.48 (m, 1H), 4.06 (d, J = 13.6 Hz, 1H), 4.24 (d, J =10.0 Hz, 1H), 4.68-4.76 (m, 1H), 5.23 (s, 1H), 5.34 (d, J = 13.6 Hz,1H), 5.78 (d, J = 7.6 Hz, 1H), 6.68 (d, J = 7.6 Hz, 1H), 6.84 (t, J =7.6 Hz, 1H), 6.95-7.00 (m, 1H), 7.03-7.15 (m, 4H). III-40

1H-NMR (CDCl3) δ: 0.94 (3H, d, J = 7.2 Hz), 1.45-1.86 (5H, m), 1.86-2.12(1H, m), 2.79 (1H, dd, J = 13.3, 3.5 Hz), 4.05 (1H, d, J = 13.7 Hz),4.27 (1H, dd, J = 11.6, 2.4 Hz), 4.56 (1H, d, J = 13.2 Hz), 5.36 (1H,dd, J = 13.6, 2.4 Hz), 5.20 (1H, s), 5.79 (1H, d, J = 7.7 Hz), 6.69 (1H,d, J = 7.4 Hz), 6.81- 6.87 (1H, m), 6.95-7.01 (1H, m), 7.05- 7.14 (4H,m). III-41

1H-NMR (CDCl3) δ: 0.96 (3H, d, J = 6.5 Hz), 1.16-1.20 (1H, m), 1.34-1.40(1H, m), 1.64-1.79 (3H, m), 1.85-1.89 (1H, m), 2.52 (1H, td, J = 13.1,2.6 Hz), 4.05 (1H, d, J = 13.8 Hz), 4.28 (1H, dd, J = 11.5, 2.2 Hz),4.70 (1H, dd, J = 13.3. 3.6 Hz), 5.23 (1H, s), 5.36 (1H, dd, J = 13.7,2.4 Hz), 5.79 (1H, d, J = 7.8 Hz), 6.68 (1H, d, J = 7.5 Hz), 6.82-6.86(1H, m), 6.98 (1H, dd, J = 8.3, 5.3 Hz), 7.02-7.15 (4H, m).

TABLE 5 III-43

1H-NMR (CDCl3) δ: 1.55 (1H, ddd, J = 26.3, 13.0, 4.6 Hz), 1.74 (1H, q, J= 12.3 Hz), 1.89 (1H, d, J = 13.1 Hz), 2.09 (1H, d, J = 12.7 Hz), 2.58(1H, td, J = 13.2, 2.6 Hz), 2.40-2.52 (1H, m), 3.54 (1H, d, J = 13.4Hz), 4.35 (1H, dd, J = 11.7, 2.3 Hz), 4.84 (1H, dd, J = 13.4, 3.8 Hz),5.23 (1H, s), 5.57 (1H, d, J = 13.4 Hz), 5.80 (1H, d, J = 7.7 Hz), 6.69(1H, d, J = 7.7 Hz), 6.82-6.86 (1H, m), 6.98 (1H, td, J = 8.2, 2.6 Hz),7.07-7.14 (4H, m), 7.20 (1H, dd, J = 8.3, 5.5 Hz). III-44

1H-NMR(CDCl3) δ: 1.83-2.00 (m, 1H), 2.08-2.23 (m, 2H), 2.37 (t, J = 13.6Hz, 1H), 2.74 (t, J = 13.2 Hz, 1H), 3.63 (d, J = 13.6 Hz, 1H), 4.51 (d,J = 11.6 Hz, 1H), 4.76-4.84 (m, 1H), 5.54 (d, J = 13.2 Hz, 1H), 5.79 (d,J = 8.0 Hz, 1H), 5.87 (s, 1H), 6.77 (d, J = 7.2 Hz, 1H), 6.85 (t, J =7.2 Hz, 1H), 7.04-7.18 (m, 5H), 7.35-7.43 (m, 1H). III-45

1H-NMR(CDCl3) δ: 0.82 (s, 3H), 0.96 (s, 3H), 1.30-1.61 (m, 4H), 2.71 (t,J = 13.2 Hz, 1H), 1.99 (d, J = 12.8 Hz, 1H), 2.54 (t, J = 12.8 Hz, 1H),4.04 (d, J = 13.6 Hz, 1H), 4.27 (dd, J = 2.0 Hz, 11.2 Hz, 1H), 4.69-4.74(m, 1H), 5.23 (s, 1H), 5.35 (dd, J = 2.4 Hz, 13.6 Hz, 1H), 5.77 (d, J =7.6 Hz, 1H), 6.68 (d, J = 7.6 Hz, 1H), 6.80-6.86 (m, 1H), 6.95-7.00 (m,1H), 7.03-7.14 (m, 4H). III-46

1H-NMR(CDCl3) δ: 1.83-2.00 (m, 1H), 2.07-2.27 (m, 2H), 2.37 (t, J = 13.2Hz, 1H), 2.67 (t, J = 13.2 Hz, 1H), 3.54 (d, J = 13.2 Hz, 1H), 4.51 (d,J = 11.2 Hz, 1H), 4.75-4.82 (m, 1H), 5.24 (s, 1H), 5.50 (d, J = 13.2 Hz,1H), 5.77 (d, J = 7.2 Hz, 1H), 6.68 (d, J = 7.6 Hz, 1H), 6.80-6.86 (m,1H), 6.95-7.02 (m, 1H), 7.05-7.14 (m, 4H), 7.16-7.23 (m, 1H) III-47

1H-NMR(CDCl3) δ: 0.82 (s, 3H), 0.97 (s, 3H), 1.24-1.44 (m, 2H),1.46-1.60 (m, 2H), 2.58-2.68 (m, 1H), 3.50 (d, J = 13.2 Hz, 1H), 4.44(dd, J = 2.8 Hz, 11.6 Hz, 1H), 4.57 (dd, J = 2.8 Hz, 13.2 Hz, 1H), 5.23(s, 1H), 5.58 (d, J = 13.6 Hz, 1H), 5.78 (d, J = 7.6 Hz, 1H), 6.68 (d, J= 7.6 Hz, 1H), 6.80- 6.86 (m, 1H), 6.95-7.03 (m, 2H), 7.05- 7.13 (m,3H), 7.18-7.24 (m, 1H). III-48

1H-NMR(CDCl3) δ: 0.10-0.16 (m, 1H), 0.25-0.31 (m, 1H), 0.36-0.49 (m,2H), 0.79 (d, J = 14.0 Hz, 1H), 0.99 (d, J = 12.8 Hz, 1H), 1.92-2.03 (m,1H), 2.18 (t, J = 12.0 Hz, 1H), 2.65-2.77 (m, 1H), 3.58 (d, J = 13.6 Hz,1H), 4.45 (dd, J = 2.4 Hz, 11.6 Hz, 1H), 4.73 (dd, J = 3.6 Hz, 13.2 Hz,1H), 5.58 (d, J = 13.6 Hz, 1H), 5.81 (d, J = 7.6 Hz, 1H), 5.88 (s, 1H),6.78 (d, J = 7.2 Hz, 1H), 6.81-6.88 (m, 1H), 7.05-7.16 (m, 5H),7.34-7.43 (m, 1 H).

TABLE 6 III-49

1H-NMR (CDCl3) δ: 0.95 (d, J = 6.5 Hz, 3H), 1.12-1.24 (m, 1H), 1.36 (dd,J = 24.1, 11.7 Hz, 1H), 1.48-1.75 (m, 2H), 1.86 (d, J = 12.7 Hz, 1H),2.59 (td, J = 13.1, 2.8 Hz, 1H), 3.59 (d, J = 13.3 Hz, 1H), 4.28 (dd, J= 11.5, 2.4 Hz, 1H), 4.73 (dd, J = 13.6, 3.0 Hz, 1H), 5.66 (d, J = 13.3Hz, 1H), 5.79 (d, J = 7.7 Hz, 1H), 5.85 (s, 1H), 6.77-6.79 (m, 1H),6.82-6.86 (m, 1H), 7.03-7.11 (m, 3H), 7.14 (d, J = 7.7 Hz, 2H), 7.36(td, J = 8.0, 5.5 Hz, 1H). III-50

1H-NMR (CDCl3) δ: 0.95 (d, J = 6.5 Hz, 3H), 1.12-1.28 (m, 1H), 1.36 (q,J = 12.0 Hz, 1H), 1.63-1.78 (m, 3H), 1.86 (d, J = 12.8 Hz, 1H), 2.52(td, J = 13.1, 2.8 Hz, 1H), 3.51 (d, J = 13.4 Hz, 1H), 4.28 (dd, J =11.6, 2.3 Hz, 1H), 4.69 (dd, J = 13.5, 3.3 Hz, 1H), 5.22 (s. 1H), 5.62(d, J = 13.4 Hz, 1H), 5.78 (d, J = 7.7 Hz, 1H), 6.68 (d, J = 7.7 Hz,1H), 6.81-6.85 (m, 1H), 6.97 (td, J = 8.3, 2.6 Hz, 1H), 7.05-7.10 (m,4H), 7.20 (dd, J = 8.4, 5.4 Hz, 1H). III-51

1H-NMR (CDCl3) δ: 1.17 (d, J = 6.1 Hz, 3H), 2.61 (dd, J = 13.3, 10.7 Hz,1H), 3.54-3.59 (m, 1H), 3.64 (t, J = 10.6 Hz, 1H), 3.96 (dd, J = 11.1,2.9 Hz, 1H), 4.07 (d, J = 13.8 Hz, 1H), 4.54 (dd, J = 10.0, 2.9 Hz, 1H),4.64 (dd, J = 13.4, 2.3 Hz, 1H), 5.26-5.30 (m, 2H), 5.75 (d, J = 7.7 Hz,1H), 6.68 (d, J = 7.7 Hz, 1H), 6.85 (t, J = 7.2 Hz, 1H), 6.98-7.03 (m,2H), 7.07-7.15 (m, 3H). III-52

1H-NMR (CDCl3) δ: 1.16 (d, J = 6.0 Hz, 3H), 2.55-2.65 (m, 1H), 3.48-3.60(m, 2H), 3.64 (t, J = 10.4 Hz, 1H), 3.94 (dd, J = 2.8 Hz, 11.2 Hz, 1H),4.54 (dd, J = 2.8 Hz, 10.0 Hz, 1H), 4.62 (dd, J = 2.0 Hz, 13.6 Hz, 1 H),5.25 (s, 1H), 5.54 (d, J = 13.2 Hz, 1H), 5.74 (d, J = 7.2 Hz, 1H), 6.68(d, J = 7.2 Hz, 1H), 6.79-6.86 (m, 1H), 6.96-7.05 (m, 2H), 7.05- 7.15(m, 3H), 7.17-7.24 (m, 1H).

TABLE 7 III-53

1H-NMR (CDCl3) δ: 1.45-1.74 (m, 4H), 1.85 (d, J = 12.0 Hz, 1H),1.95-2.02 (m, 1H), 2.61 (t, J = 12.4 Hz, 1H), 3.58 (d, J = 14.0 Hz, 1H),4.27 (d, J = 10.8 Hz, 1H), 4.74 (d, J = 12.4 Hz, 1H), 5.65 (d, J = 14.0Hz, 1H), 5.78 (d, J = 6.8 Hz, 1H), 5.85 (s, 1H), 6.75-6.88 (m, 2H),7.02-7.15 (m, 5H), 7.34-7.40 (m, 1H). III-55

1H-NMR (CDCl3) δ: 0.12-0.18 (m, 1H), 0.25-0.31 (m, 1H), 0.36-0.49 (m,2H), 0.78 d, J = 14.0 Hz, 1H), 0.99 (d, J = 12.4 Hz, 1H), 1.92-2.00 (m,1H), 2.18 (t, J = 11.6 Hz, 1H), 2.58-2.68 (m, 1H), 3.48 (d, J = 13.2 Hz,1H), 4.44 (dd, J = 2.0 Hz, 11.6 Hz, 1H), 4.70 (dd, J = 3.2 Hz, 12.8 Hz,1H), 5.24 (s, 1H), 5.53 (d, J = 13.6 Hz, 1H), 5.77 (d, J = 8.0 Hz, 1H),6.89 (d, J = 7.2 Hz, 1H), 6.80-6.87 (m, 1H), 6.95-7.02 (m, 2H),7.03-7.14 (m, 3H), 7.20-7.26 (m, 1H). III-57

1H-NMR δ: 7.20 (dd, J = 8.6, 5.5 Hz, 1H), 7.14-7.08 (m, 3H), 7.03-6.97(m, 2H), 6.85-6.82 (m, 1H), 6.68 (d, J = 7.7 Hz, 1H), 5.81 (d, J = 7.5Hz, 1H), 5.53 (d, J = 13.6 Hz, 1H), 5.21 (s, 1H), 4.69-4.63 (m, 1H),3.54 (d, J = 13.6 Hz, 1H), 2.85-2.80 (m, 1H), 2.66 (brs, 1H), 2.15-2.00(m, 2H), 1.95-1.80 (m, 2H) III-58

1H-NMR (CDCl3) δ: 0.90 (d, J = 6.5 Hz, 3H), 1.23 (ddd, J = 25.6, 12.8,4.1 Hz, 1H), 1.63-1.86 (m, 3H), 1.95 (d, J = 13.7 Hz, 1H), 2.17 (t, J =12.3 Hz, 1H), 3.51 (d, J = 13.4 Hz, 1H), 4.25 (d, J = 11.0 Hz, 1H), 4.60(d, J = 12.0 Hz, 1H), 5.21 (s, 1H), 5.61 (d, J = 13.3 Hz, 1H), 5.78 (d,J = 7.7 Hz, 1H), 6.68 (d, J = 7.8 Hz, 1H), 6.83 (t, J = 6.7 Hz, 1H),6.99 (t, J = 8.2 Hz, 1H), 7.05-7.09 (m, 4H), 7.20 (dd, J = 8.1, 5.7 Hz,1H). III-59

1H-NMR (CDCl3) δ: 1.45-1.79 (m, 4H), 1.87 (d, J = 10.8 Hz, 1H), 1.99 (d,J = 12.8 Hz, 1H), 2.54 (t, J = 12.8 Hz, 1H), 4.04 (d, J = 13.6 Hz, 1H),4.27 (dd, J = 2.0 Hz, 11.2 Hz, 1H), 4.69-4.74 (m, 1H), 5.23 (s, 1H),5.35 (dd, J = 2.4 Hz, 13.6 Hz, 1H), 5.77 (d, J = 7.6 Hz, 1H), 6.68 (d, J= 7.6 Hz, 1H), 6.80-6.86 (m, 1H), 6.95-7.00 (m, 1H), 7.03-7.14 (m, 4H).

To Compound III-2 (4.0 g, 8.3 mmol) were added potassium carbonate(1483.4 mg, 10.7 mmol), potassium iodide (549.5 mg, 3.3 mmol),tetrahydrofuran (33.1 g), N,N-dimethylacetamide (3.8 g), and water (80.3mg), and the mixture was stirred. The temperature was increased to 60°C., and chloromethyl methyl carbonate (1758.9 mg, 14.2 mmol) was added.The mixture was stirred at 60° C. for 9 hours and cooled to 20° C.Acetic acid (822.0 mg), 2-propanol (3.1 g), and water (20.0 g) wereadded, and the mixture was extracted twice with tetrahydrofuran (1.8 g,8.9 g). The obtained organic layer was concentrated under reducedpressure to a liquid weight of about 32 g by distilling off the solvent.The temperature was increased to 45° C., 2-propanol (1.6 g) was added,and the mixture was cooled to 20° C. An aqueous solution of sodiumacetate prepared from sodium acetate (339.0 mg) and water (46.0 g) wasadded, and the mixture was cooled to 5° C. The mixture was stirred at 5°C. for 3 hours, and the produced pale yellowish white precipitates werefiltered. The obtained solids were washed with a mixed solution of2-propanol (4.7 g) and water (6.0 g), and the solids were washed with2-propanol (6.3 g) again. Dimethylsulfoxide (30.9 g) was added to theobtained pale yellowish white solids, and the mixture was stirred. Thetemperature was increased to 60° C., and a mixed solution ofdimethylsulfoxide (2.2 g) and water (4.8 g) was added. Further, a mixedsolution of dimethylsulfoxide (19.9 g) and water (28.4 g) was added, andthe mixture was cooled to 20° C. The mixture was stirred at 20° C. for 3hours, and the produced white precipitates were filtered. The obtainedsolids were washed with a mixed solution of dimethylsulfoxide (8.0 g)and water (4.8 g), and the solids were washed with water (12.0 g) again.The obtained solids were dried to obtain white crystals (1-form) ofCompound II-6 (4.21 g).

¹H-NMR (DMSO-D6) δ: 2.91-2.98 (1H, m), 3.24-3.31 (1H, m), 3.44 (1H, t,J=10.4 Hz), 3.69 (1H, dd, J=11.5, 2.8 Hz), 3.73 (3H, s), 4.00 (1H, dd,J=10.8, 2.9 Hz), 4.06 (1H, d, J=14.3 Hz), 4.40 (1H, d, J=11.8 Hz), 4.45(1H, dd, J=9.9, 2.9 Hz), 5.42 (1H, dd, J=14.4, 1.8 Hz), 5.67 (1H, d,J=6.5 Hz), 5.72-5.75 (3H, m), 6.83-6.87 (1H, m), 7.01 (1H, d, J=6.9 Hz),7.09 (1H, dd, J=8.0, 1.1 Hz), 7.14-7.18 (1H, m), 7.23 (1H, d, J=7.8 Hz),7.37-7.44 (2H, m).

Powder X-ray diffraction 2θ(°): 8.6±0.2°, 14.1±0.2°, 17.4±0.2°,20.0±0.2°, 24.0±0.2°, 26.3±0.2°, 29.6±0.2°, and 35.4±0.2°.

FIG. 3 shows powder X-ray diffraction results of I-form crystals ofCompound II-6.

Example 11: Method for Producing Compound II-61

First Step

To a solution of chloromethyl chloroformate (300 mg, 2.33 mmol) andCompound 73 (330 mg, 2.79 mmol) in dichloromethane (6.0 mL) was addedpyridine (207 μL, 2.56 mmol) at 0° C. under nitrogen atmosphere, and themixture was stirred at 0° C. for 30 minutes, was warmed up to roomtemperature and was stirred for 1 hour. To the mixture was added 2 mol/Laqueous solution of hydrochloric acid and the mixture was extracted withdichloromethane. The obtained organic layer was washed with brine, driedover anhydrous magnesium sulfate, and concentrated under reducedpressure to obtain Compound 74 (440 mg, 90%).

1H-NMR (CDCl3) δ:1.65 (s, 6H), 3.77 (s, 3H), 5.71 (s, 2H).

Second Step

Compound III-2 (300 mg, 0.62 mmol), potassium carbonate (172 mg, 1.24mmol), potassium iodide (103 mg, 0.62 mmol) and Compound 74 (261 mg,1.24 mmol) were dissolved in DMA (3.0 mL) and the mixture was stirred at80° C. for 3 hours. To the mixture was added 2 mol/L aqueous solution ofhydrochloric acid and the mixture was extracted with ethyl acetate. Theobtained organic layer was washed with brine, dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure. The obtainedresidue was purified by silica gel column chromatography(chloroform-methanol) to obtain Compound II-61 (360 mg, 86%).

1H-NMR (CDCl3) δ: 1.63 (s, 3H), 1.67 (s, 3H), 2.86-2.93 (m, 1H),3.38-3.61 (m, 2H), 3.68-3.78 (m, 4H), 3.90-3.96 (m, 1H), 4.06 (d, J=14.0Hz, 1H), 4.51 (dd, J=2.0 Hz, 9.6 Hz, 1H), 4.65 (d, J=12.4 Hz, 1H), 5.21(d, J=14.4 Hz, 1H), 5.36 (s, 1H), 5.80-5.95 (m. 3H), 6.85-6.92 (m, 2H),7.03-7.22 (m, 6H).

Example 12: Method for Producing Compound II-4

To a solution of Compound III-2 (90 mg, 0.186 mmol) in dichloromethane(2 mL) were added acetic anhydride (0.053 mL, 0.558 mmol), triethylamine(0.077 mL, 0.558 mmol) and a catalytic amount of DMAP, and the mixturewas stirred at room temperature for 2 hours. The mixture wasconcentrated under reduced pressure and the obtained residue waspurified by silica gel column chromatography (chloroform-methanol). Tothe obtained solution was added ether and the precipitated solid wasfiltered to obtain Compound II-4 (71 mg, 73%).

1H-NMR (CDCl3) δ:2.46 (s, 3H), 2.88-2.99 (m, 1H), 3.35-3.50 (m, 1H),3.60-3.65 (m, 1H), 3.75-3.83 (m, 1H), 3.90-4.00 (m, 1H), 4.05 (d, J=14.0Hz, 1H), 4.52-4.57 (m, 1H), 4.60-4.70 (m, 1H), 5.24-5.34 (m, 1H), 5.35(s, 1H), 5.88 (d, J=7.6 Hz, 1H), 6.85-6.82 (m, 1H), 6.90-7.05 (m, 2H),7.06-7.20 (m, 4H)

LC/MS (ESI): m/z=526.2 [M+H]⁺, RT=1.87 min, method (1)

Example 13: Method for Producing Compound II-65

First Step

To a solution of triphosgene (300 mg, 2.54 mmol) in dichloromethane (6.0mL) was added pyridine (257 μL, 3.17 mmol) at 0° C. under nitrogenatmosphere and the mixture was stirred for 15 minutes. To the mixturewas added a solution of Compound 73 (377 mg, 1.27 mmol) indichloromethane (1.0 mL), and the mixture was stirred at 0° C. for 15minutes, warmed up to room temperature and stirred for 15 minutes. Themixture was concentrated under reduced pressure, ethyl acetate (4.0 mL)was added thereto, and the mixture was filtered. The filtrate wasconcentrated under reduced pressure to obtain Compound 75 (380 mg).

Second Step

To a solution of Compound III-2 (350 mg, 0.724 mmol) in dichloromethane(3.5 mL) were added Compound 75 (196 mg, 1.09 mmol) and triethylamine(301 μL, 2.17 mmol) at 0° C. and the mixture was stirred at 0° C. for 30minutes. To the mixture was added 2 mol/L aqueous solution ofhydrochloric acid and the mixture was extracted with dichloromethane.The obtained organic layer was washed with brine, dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure. The obtainedresidue was purified by silica gel column chromatography(chloroform-methanol) to obtain Compound II-65 (380 mg, 84%).

1H-NMR (CDCl3) δ:1.73 (s, 3H), 1.77 (s, 3H), 2.90-2.99 (m, 1H),3.37-3.43 (m, 1H), 3.57 (t, J=8.8 Hz, 1H), 3.76 (dd, J=2.8 Hz, 12.0 Hz,1H), 3.81 (s, 3H), 3.94 (dd, J=2.8 Hz, 10.8 Hz, 1H), 4.05 (d, J=14.0 Hz,1H), 4.55 (dd, J=2.8 Hz, 9.6 Hz, 1H), 4.65 (d, J=12.0 Hz, 1H), 5.28 (d,J=12.0 Hz, 1H), 5.34 (s, 1H), 5.89 (d, J=8.0 Hz, 1H), 6.86-6.95 (m, 2H),7.03-7.15 (m, 5H).

Example 14: Method for Producing Compound II-129

To a solution of Compound 76 (276 mg, 0.402 mmol) in THF (1 mL) wereadded acetic acid (121 mg, 2.01 mmol) and 1 mol/L TBAF in THF (1.21 mL,1.21 mmol) under ice-water bath and the mixture was stirred at roomtemperature for 4 hours. The mixture was concentrated under reducedpressure. The obtained residue was purified by silica gel columnchromatography (ethyl acetate-methanol) to obtain Compound II-129 (179mg, 78%).

LC/MS (ESI): m/z=572.0 [M+H]⁺, RT=1.74 min, method (2)

Example 15: Method for Producing Compound II-115

To a solution of Compound III-2 (300 mg, 0.62 mmol) in DMF (4 mL) wereadded potassium carbonate (258 mg, 1.87 mmol), 4-(chloromethyl)phenylacetate (344 mg, 1.87 mmol) and sodium iodide (139 mg, 1.87 mmol) atroom temperature and the mixture was stirred at 65° C. for 1 hour. Tothe mixture was added water and the mixture was extracted with ethylacetate. The obtained organic layer was washed with water, dried overanhydrous sodium sulfate, and concentrated under reduced pressure. Theobtained residue was purified by silica gel column chromatography (ethylacetate-methanol) to obtain Compound II-115 (120 mg, 31%).

LC/MS (ESI): m/z=631.95 [M+H]⁺, RT=2.07 min, method (2)

Example 16: Method for Producing Compound II-143

To a solution of Compound III-2 (150 mg, 0.31 mmol) in dichloromethane(2 mL) 3 mmol/g triphenylphosphine supported on polymer (310 mg, 0.93mmol), pyridin-4-ylmethanol (68 mg, 0.62 mmol) and 40% DEAD in toluene(270 mg, 0.62 mmol) at room temperature and the mixture was stirred atroom temperature for 30 minutes. The mixture was purified by aminocolumn chromatography (ethyl acetate-methanol) to obtain Compound II-143(63 mg, 35%).

LC/MS (ESI): m/z=575.00 [M+H]⁺, RT=1.43 min, method (2)

Example 17: Method for Producing Compound II-27

To a solution of Compound III-2 (65 mg, 0.134 mmol) in pyridine (0.8 mL)was added dimethylcarbamoyl chloride (21.7 mg, 0.202 mmol) and themixture was stirred at 80° C. over night. To the mixture was added 1mol/L aqueous solution of hydrochloric acid and the mixture wasextracted with ethyl acetate. The obtained organic layer was washed withbrine, dried over anhydrous magnesium sulfate, and concentrated underreduced pressure. The obtained residue was solidified with ethylacetate-hexane to obtain Compound II-27 (65 mg, 87%).

1H-NMR (CDCl3) δ:2.89 (t, J=11.2 Hz, 1H), 2.99 (s, 1H), 3.01 (s, 3H),3.18-3.26 (m, 4H), 3.45 (t, J=10.8 Hz, 1H), 3.59 (t, J=10.8 Hz, 1H),3.70-3.80 (m, 1H), 3.90-3.98 (m, 1H), 4.03 (d, J=13.6 Hz, 1H), 4.50-4.70(m, 2H), 5.21-5.35 (m, 2H), 5.82 (d, J=7.6 Hz, 1H), 6.91 (t, J=7.6 Hz,1H), 7.00-7.20 (m, 6H).

Example 18: Method for Producing Compound II-55

To a solution of ethyl phosphorodichloridate (135 mg, 0.829 mmol) indichloromethane (3 mL) was added L-valine methyl ester hydrochloride(139 mg, 0.829 mmol) and then added dropwise a solution of triethylamine(168 mg, 1.66 mmol) in dichloromethane (2 mL) at −78° C. The mixture wasstirred at room temperature for 1 hour. Compound III-2 (200 mg, 0.414mmol) and triethylamine (126 mg, 1.25 mmol) were added thereto, and themixture was stirred at same temperature for 6 hours. The mixture wasconcentrated and the obtained residue was purified by silica gel columnchromatography (ethyl acetate-methanol) to obtain Compound II-55 (112mg, 38%).

LC/MS (ESI): m/z=705.05 [M+H]⁺, RT=2.18 min, method (2)

Example 19: Method for Producing Compound II-57

To a solution of ethyl phosphorodichloridate (202 mg, 1.24 mmol) indichloromethane (3 mL) was added dropwise a mixture of triethylamine(126 mg, 1.24 mmol) and methyl glycolate (112 mg, 1.24 mmol) indichloromethane (2 mL). The mixture was stirred at room temperature for2 hours. Compound III-2 (200 mg, 0.414 mmol) and triethylamine (126 mg,1.25 mmol) were added thereto and the mixture was stirred at sametemperature for 1 hour. The mixture was concentrated and the obtainedresidue was purified by silica gel column chromatography (ethylacetate-methanol) to obtain Compound II-57 (143 mg, 52%).

LC/MS (ESI): m/z=664.00 [M+H]⁺, RT=1.93 min, method (2)

Example 20: Method for Producing Compound II-58

To a solution of phosphoryl chloride (1.53 g, 10 mmol) indichloromethane (10 mL) was added dropwise the mixture of triethylamine(2.12 g, 20.95 mmol) and methyl glycolate (1.89 mg, 21 mmol) indichloromethane (5 mL). The mixture was stirred at room temperature for2 hours. To the mixture (2 mL) were added Compound III-2 (200 mg, 0.414mmol) and triethylamine (126 mg, 1.25 mmol) and the mixture was stirredat same temperature for 1 hour. The mixture was concentrated and theobtained residue was purified by silica gel column chromatography (ethylacetate-methanol) to obtain Compound II-58 (166 mg, 57%).

LC/MS (ESI): m/z=707.90 [M+H]⁺, RT=1.93 min, method (2)

The following example compounds were synthesized from commerciallyavailable compounds or intermediates suitably synthesized fromcommercially available compounds according to the above examples.

TABLE 8 No. Structure NMR or LC/MS II-5

1H-NMR (DMSO-d6) δ: 2.04 (s, 3H), 2.90-3.00 (m, 1H), 3.44-3.50 (m, 2H),3.64-3.72 (m, 1H), 3.95-4.00 (m, 1H), 4.11-4.10 (m, 1H), 4.20-4.30 (m,2H), 5.40- 5.5.46 (m, 1H), 6.62-5.75 (m, 4H), 6.80-6.90 (m, 1H),6.98-7.10 (m, 1H), 7.11-7.20 (m, 2H), 7.21-7.30 (m, 1H), 7.45-7.50 (m,2H) II-7

1H-NMR (CDCl3) δ: 2.85-2.97 (m, 1H), 3.38 (s, 3H), 3.39-3.48 (m, 1H),3.54 (t, J = 10.4 Hz, 1H), 3.68 (t, J = 4.4 Hz, 2H), 3.74 (dd, J = 2.8Hz, 12.0 Hz, 1H), 3.92 (dd, J = 2.8 Hz, 10.8 Hz, 1H), 4.05 (d, J = 13.6Hz, 1H), 4.36 (q, J = 4.4 Hz, 2H), 4.51 (dd, J = 2.8 Hz, 9.6 Hz, 1H),4.65 (d, J = 12.0 Hz, 1H), 5.27 (dd, J = 2.0 Hz, 13.6 Hz, 1H), 5.34 (s,1H), 5.86 (d, J = 8.0 Hz, 1H), 5.93 (s, 2H), 6.81-6.89 (m, 2H), 6.98-7.15 (m, 5H). II-8

LC/MS (ESI): m/z = 508 [M + H]+, RT = 1.76 min, method (2) II-9

1H-NMR (CDCl3) δ: 2.05 (s, 3H), 2.92-3.02 (m, 1H), 3.40-3.48 (m, 1H),3.51-3.62 (m, 2H), 3.72-3.80 (m, 1H), 3.88-3.92 (m, 1H), 4.50-4.56 (m,1H), 4.64-4.72 (m, 1H), 5.55 (d, J = 13.6 Hz, 1H), 5.78-5.82 (m, 1H),5.84-5.88 (m, 1H), 5.90-5.98 (m, 2H), 6.82-7.00 (m, 2H), 7.00-7.20 (m,5H), 7.35-7.42 (m, 1H) II-10

LC/MS (ESI): m/z = 554 [M + H]+, RT = 1.76 min, method (1) II-11

LC/MS (ESI): m/z = 598 [M + H]+, RT = 1.80 min, method (2)

TABLE 9 No. Structure NMR or LC/MS II-16

LC/MS (ESI): m/z = 508 [M + H]+, RT = 1.76 min, method (2) II-17

LC/MS (ESI): m/z = 538 [M + H]+, RT = 1.78 min, method (2) II-18

LC/MS (ESI): m/z = 554 [M + H]+, RT = 1.81 min, method (2) II-19

LC/MS (ESI): m/z = 598 [M + H]+, RT = 1.85 min, method (2) II-24

1H-NMR (CDCl3) δ: 1.33 (3H, t, J = 7.0 Hz), 2.82 (2H, d, J = 6.1 Hz),2.93 (1H, t, J = 11.2 Hz), 3.42 (1H, t, J =11.4 Hz), 3.59 (1H, t, J =10.2 Hz), 3.78 (1H, d, J = 11.2 Hz), 3.96 (1H, d, J = 10.3 Hz), 4.06(1H, d, J = 13.8 Hz), 4.55 (1H, d, J = 8.9 Hz), 4.63 (1H. d, J = 13.6Hz), 5.29 (1H, d, J = 13.9 Hz), 5.36 (1H, s), 5.88 (1H, d, J = 7.4 Hz),6.90 (1H, s), 7.03- 7.12 (6H, m). II-25

1H-NMR (CDCl3) δ: 1.42 (d, J = 6.8 Hz, 6H), 2.85- 3.05 (m, 2H),3.40-3.49 (m, 1H), 3.59 (t, J = 10.4 Hz, 1H), 3.76 (d, J = 11.4 Hz, 1H),3.94 (d, J = 10.4 Hz, 1H), 4.06 (d, J = 14.1 Hz, 1H), 4.51-4.57 (m, 1H),4.59-4.70 (m, 1H), 5.25-5.32 (m, 1H), 5.35- 5.39 (m, 1H), 5.80-5.89 (m,1H), 6.85-7.15 (m, 7H).

TABLE 10 No. Structure NMR or LC/MS II-26

LC/MS (ESI): m/z = 542 [M + H]+, RT = 1.92 min, method (1) II-28

LC/MS (ESI): m/z = 610 [M + H]+, RT = 1.57 min, method (1) II-29

LC/MS (ESI): m/z = 554 [M + H]+, RT = 2.10 min, method (1) II-30

LC/MS (ESI): m/z = 568 [M + H]+, RT = 1.91 min, method (1) II-31

1H-NMR (CDCl3) δ: 1.42 (d, J = 6.8 Hz, 6H), 2.90-3.07 (m, 2H), 3.44 (t,J = 10.8 Hz, 1H), 3.60 (d, J = 12.8 Hz, 2H), 3.77 (d, J = 10.8 Hz, 1H),3.93 (dd, J = 10.8, 2.8 Hz, 1H), 4.56 (dd, J = 9.6, 2.8 Hz, 1H), 4.67(m, 1H), 5.59 (m, 1H), 5.87 (m, 1H), 5.59 (s, 1H), 6.91-7.21 (m, 7H),7.38 (m, 1H).

TABLE 11 No. Structure NMR or LC/MS II-32

1H-NMR (CDCl3) δ: 2.88 (1H, t, J = 11.2 Hz), 3.28- 3.39 (2H, m), 3.72(1H, d, J = 12.6 Hz), 3.86 (1H, d, J = 9.6 Hz), 4.03 (1H, d, J = 13.9Hz), 4.45 (1H, d, J = 8.6 Hz), 4.67 (1H, d, J = 13.1 Hz), 5.19-5.26 (2H,m), 5.45 (1H, d, J = 10.9 Hz), 5.63 (1H, d, J = 10.9 Hz), 5.77 (1H, d, J= 7.6 Hz), 6.40 (1H, d, J = 7.8 Hz), 6.68 (1H, t, J = 6.9 Hz), 6.94-7.01(2H, m), 7.03-7.12 (3H, m), 7.29-7.38 (3H, m), 7.61 (2H, d, J = 7.1 Hz).II-33

1H-NMR (CDCl3) δ: 1.46 (t, J = 7.2 Hz, 3H), 2.95 (m, 1H), 3.42 (td, J =12.0, 2.4 Hz, 1H), 3.58 (t, J = 10.4 Hz, 1H), 3.78 (dd, J = 12.0, 2.8Hz, 1H), 3.95 (dd, J = 11.2, 2.8 Hz, 1H), 4.07 (d, J = 13.6 Hz, 1H),4.41 (m, 2H), 4.56 (dd, J = 10.0, 2.8 Hz, 1H), 4.67 (dd, J = 10.0, 2.4Hz, 1H), 5.29 (dd, J = 2.0 Hz, 1H), 5.36 (s, 1H), 5.91 (d, J = 8.0 Hz,1H), 6.88-7.15 (m, 7H). II-34

1H-NMR (CDCl3) δ: 1.46 (m, 6H), 2.95 (m, 1H), 3.41 (td, J = 12.0, 2.0Hz, 1H), 3.58 (t, J = 10.8 Hz, 1H), 3.77 (dd, J = 12.0, 3.2 Hz, 1H),3.95 (dd, J = 10.8, 2.4 Hz, 1H), 4.06 (d, J = 14.0 Hz, 1H), 4.55 (dd, J= 9.6, 2.8 Hz, 1H), 4.67 (d, J = 13.6 Hz, 1H), 5.04 (m, 1H), 5.29 (d, J= 13.6 Hz, 1H), 5.36 (s, 1H), 5.90 (d, J = 8.0 Hz, 1H), 6.90-7.13 (m,7H). II-36

LC/MS (ESI): m/z = 663 [M + H]+, RT = 2.29 min, method (1) II-37

LC/MS (ESI): m/z = 626 [M + H]+, RT = 2.18 min, method (1)

TABLE 12 No. Structure NMR or LC/MS II-38

LC/MS (ESI): m/z = 570 [M + H]+, RT = 1.85 min, method (2) II-39

LC/MS (ESI): m/z = 606 [M + H]+, RT = 2.12 min, method (2) II-40

LC/MS (ESI): m/z = 568 [M + H]+, RT = 1.92 min, method (2) II-41

LC/MS (ESI): m/z = 598 [M + H]+, RT = 2.27 min, method (2) II-42

LC/MS (ESI): m/z = 638 [M + H]+, RT = 2.17 min, method (2)

TABLE 13 No. Structure NMR or LC/MS II-43

LC/MS (ESI): m/z = 584 [M + H]+, RT = 2.18 min, method (2) II-44

LC/MS (ESI): m/z = 588 [M + H]+, RT = 2.00 min, method (2) II-45

LC/MS (ESI): m/z = 580 [M + H]+, RT = 2.14 min, method (2) II-46

LC/MS (ESI): m/z = 588 [M + H]+, RT = 2.04 min, method (2) II-47

LC/MS (ESI): m/z = 580 [M + H]+, RT = 2.17 min, method (2)

TABLE 14 No. Structure NMR or LC/MS II-48

LC/MS (ESI): m/z = 586 [M + H]+, RT = 2.03 min, method (2) II-49

LC/MS (ESI): m/z = 596 [M + H]+, RT = 2.18 min, method (2) II-50

LC/MS (ESI): m/z = 566 [M + H]+, RT = 2.02 min, method (2) II-51

LC/MS (ESI): m/z = 566 [M + H]+, RT = 2.08 min, method (2) II-52

LC/MS (ESI): m/z = 568 [M + H]+, RT = 1.93 min, method (2)

TABLE 15 No. Structure NMR or LC/MS II-53

LC/MS (ESI): m/z = 598.1 [M + H]+, RT = 1.96 min, method (2) II-54

1H-NMR (CDCl3) δ: 2.89-2.98 (m, 1H), 3.30-3.43 (m, 2H), 3.57 (d, J =13.4 Hz, 1H), 3.73 (dd, J = 11.6, 2.8 Hz, 1H), 3.87 (dd, J = 10.7, 2.4Hz, 1H), 4.49 (dd, J = 9.9, 2.5 Hz, 1H), 4.72 (d, J = 12.9 Hz, 1H), 5.43(d, J = 10.8 Hz, 1H), 5.51 (d, J = 13.4 Hz, 1H), 5.64 (d, J = 10.9 Hz,1H), 5.78 (d, J = 7.7 Hz, 1H), 5.84 (s, 1H), 6.44 (d, J = 7.8 Hz, 1H),6.67 (t, J = 7.0 Hz, 1H), 7.02-7.13 (m, 5H), 7.29-7.40 (m, 4H), 7.64 (d,J = 7.7 Hz, 2H). II-56

LC/MS (ESI): m/z = 595.90 [M + H]+, RT = 1.93 min, method (2) II-59

LC/MS (ESI): m/z = 705.05 [M + H]+, RT = 2.16 min, method (2)

TABLE 16 No. Structure NMR or LC/MS II-60

LC/MS (ESI): m/z = 691.00 [M + H]+, RT = 2.08 min, method (2) II-62

LC/MS (ESI): m/z = 615.95 [M + H]+, RT = 2.07 min, method (2) II-63

LC/MS (ESI): m/z = 579.95 [M + H]+, RT = 1.92 min, method (2) 11-64

LC/MS (ESI): m/z = 642.35 [M + H]+, RT = 2.05 min, method (2) II-66

LC/MS (ESI): m/z = 654.05 [M + H]+, RT = 2.43, 2.51 min, method (2)

TABLE 17 No. Structure NMR or LC/MS II-67

LC/MS (ESI): m/z = 600.00 [M + H]+, RT = 2.05, 2.11 min, method (2)II-68

LC/MS (ESI): m/z = 569.95 [M + H]+, RT = 1.84 min, method (2) II-69

LC/MS (ESI): m/z = 568.00 [M + H]+, RT = 2.17 min, method (2) II-70

LC/MS (ESI): m/z = 598.00 [M + H]+, RT = 2.23 min, method (2) II-71

LC/MS (ESI): m/z = 599.05 [M + H]+, RT = 1.99 min, method (2)

TABLE 18 No. Structure NMR or LC/MS II-72

LC/MS (ESI): m/z = 656.00 [M + H]+, RT = 2.13 min, method (2) II-73

LC/MS (ESI): m/z = 719.05 [M + H]+, RT = 2.28 min, method (2) II-74

LC/MS (ESI): m/z = 638.95 [M + H]+, RT = 1.89 min, method (2) II-75

LC/MS (ESI): m/z = 668.95 [M + H]+, RT = 1.97 min, method (2)

TABLE 19 No. Structure NMR or LC/MS II-76

LC/MS (ESI): m/z = 671.00 [M + H]+, RT = 2.24 min, method (2) II-77

LC/MS (ESI): m/z = 612.10 [M + H]+, RT = 2.45 min, method (2) II-78

LC/MS (ESI): m/z = 598.00 [M + H]+, RT = 2.29 min, method (2) II-79

LC/MS (ESI): m/z = 672 [M + H]+, RT = 2.27 min, method (1)

TABLE 20 No. Structure NMR or LC/MS II-80

LC/MS (ESI): m/z = 706 [M + H]+, RT = 2.39 min, method (1) II-81

LC/MS (ESI): m/z = 644 [M + H]+, RT = 2.13 min, method (1) II-82

LC/MS (ESI): m/z = 630 [M + H]+, RT = 2.03 min, method (1) II-83

LC/MS (ESI): m/z = 644 [M + H]+, RT = 2.06 min, method (1)

TABLE 21 No. Structure NMR or LC/MS II-84

LC/MS (ESI): m/z = 644 [M + H]+, RT = 2.15 min, method (1) II-85

LC/MS (ESI): m/z = 692 [M + H]+, RT = 2.31 min, method (1) II-86

LC/MS (ESI): m/z = 670 [M + H]+, RT = 2.20 min, method (1) II-87

LC/MS (ESI): m/z = 700 [M + H]+, RT = 2.45 min, method (1)

TABLE 22 No. Structure NMR or LC/MS II-88

LC/MS (ESI): m/z = 672 [M + H]+, RT = 2.31 min, method (1) II-89

LC/MS (ESI): m/z = 706 [M + H]+, RT = 2.37 min, method (1) II-90

LC/MS (ESI): m/z = 644 [M + H]+, RT = 2.13 min, method (1) II-91

LC/MS (ESI): m/z = 670 [M + H]+, RT = 2.16 min, method (1)

TABLE 23 No. Structure NMR or LC/MS II-92

LC/MS (ESI): m/z = 617.00 [M + H]+, RT = 2.09 min, method (2) II-93

LC/MS (ESI): m/z = 586.00 [M + H]+, RT = 1.91 min, method (2) II-94

LC/MS (ESI): m/z = 598.00 [M + H]+, RT = 1.89 min, method (2) II-95

LC/MS (ESI): m/z = 598.00 [M + H]+, RT = 1.89 min, method (2) II-96

LC/MS (ESI): m/z = 600.00 [M + H]+, RT = 2.01 min, method (2)

TABLE 24 No. Structure NMR or LC/MS II-97 

LC/MS (ESI): m/z = 626.00 [M + H]+, RT = 1.98 min, method (2) II-98 

LC/MS (ESI): m/z = 611.95 [M + H]+, RT = 1.93 min, method (2) II-99 

LC/MS (ESI): m/z = 626.05 [M + H]+, RT = 2.46 min, method (2) II-100

LC/MS (ESI): m/z = 682.05 [M + H]+, RT = 2.27 min, method (2)

TABLE 25 No. Structure NMR or LC/MS II-101

LC/MS (ESI): m/z = 719.05 [M + H]+, RT = 2.26 min, method (2) II-102

LC/MS (ESI): m/z = 731.15 [M + H]+, RT = 2.29 min, method (2) II-103

LC/MS (ESI): m/z = 691.10 [M + H]+, RT = 2.05 min, method (2) II-104

LC/MS (ESI): m/z = 688.95 [M + H]+, RT = 1.98 min, method (2)

TABLE 26 No. Structure NMR or LC/MS II-105

LC/MS (ESI): m/z = 759.05 [M + H]+, RT = 2.53 min, method (2) II-106

LC/MS (ESI): m/z = 639.95 [M + H]+, RT = 2.01 min, method (2) II-107

LC/MS (ESI): m/z = 683.95 [M + H]+, RT = 1.87 min, method (2) II-108

LC/MS (ESI): m/z = 625.00 [M + H]+, RT = 1.75 min, method (2)

TABLE 27 No. Structure NMR or LC/MS II-109

LC/MS (ESI): m/z = 640.00 [M + H]+, RT = 1.90 min, method (2) II-110

LC/MS (ESI): m/z = 633.90 [M + H]+, RT = 1.82 min, method (2) II-111

LC/MS (ESI): m/z = 661.00 [M + H]+, RT = 1.90 min, method (2) II-112

LC/MS (ESI): m/z = 624.95 [M + H]+, RT = 1.38 min, method (2)

TABLE 28 No. Structure NMR or LC/MS II-113

LC/MS (ESI): m/z = 691.95 [M + H]+, RT = 2.00 min, method (2) II-114

LC/MS (ESI): m/z = 604.00 [M + H]+, RT = 2.09 min, method (2) II-116

LC/MS (ESI): m/z = 631.00 [M + H]+, RT = 2.18 min, method (2) II-117

LC/MS (ESI): m/z = 620.00 [M + H]+, RT = 1.93 min, method (2)

TABLE 29 No. Structure NMR or LC/MS II-118

LC/MS (ESI): m/z = 620.00 [M + H]+, RT = 1.93 min, method (2) II-119

LC/MS (ESI): m/z = 614 [M + H]+, RT = 2.31 min, method (1) II-120

LC/MS (ESI): m/z = 614 [M + H]+, RT = 2.24 min, method (1) II-121

LC/MS (ESI): m/z = 686 [M + H]+, RT = 2.27 min, method (1)

TABLE 30 No. Structure NMR or LC/MS II-122

LC/MS (ESI): m/z = 642 [M + H]+, RT = 2.19 min, method (1) II-123

LC/MS (ESI): m/z = 642 [M + H]+, RT = 2.17 min, method (1) II-124

LC/MS (ESI): m/z = 662 [M + H]+, RT = 2.22 min, method (1) II-125

LC/MS (ESI): m/z = 668 [M + H]+, RT = 2.32 min, method (1)

TABLE 31 No. Structure NMR or LC/MS II-126

LC/MS (ESI): m/z = 587.95 [M + H]+, RT = 2.24 min, method (2) II-127

LC/MS (ESI): m/z = 588.05 [M + H]+, RT = 2.17 min, method (2) II-128

LC/MS (ESI): m/z = 686.00 [M + H]+, RT = 2.67 min, method (2) II-130

LC/MS (ESI): m/z = 645.95 [M + H]+, RT = 2.12 min, method (2)

TABLE 32 No. Structure NMR or LC/MS II-131

LC/MS (ESI): m/z = 615.00 [M + H]+, RT = 2.24 min, method (2) II-132

LC/MS (ESI): m/z = 658.95 [M + H]+, RT = 2.31 min, method (2) II-133

LC/MS (ESI): m/z = 661.00 [M + H]+, RT = 2.06 min, method (2) II-134

LC/MS (ESI): m/z = 656 [M + H]+, RT = 2.24 min, method (1)

TABLE 33 No. Structure NMR or LC/MS II-135

1H-NMR (CDCl3) δ: 1.24 (s, 3H), 1.38 (s, 3H), 2.94 (td, J = 11.8, 3.5Hz, 1H), 3.44 (dd, J = 12.0, 10.9 Hz, 1H), 3.57 (t, J = 10.9 Hz, 1H),3.78 (dd, J = 12.0, 3.5 Hz, 1H), 3.96 (dd, J = 10.9, 2.9 Hz, 1H),4.05-4.12 (m, 3H), 4.58 (dd, J = 10.0, 2.9 Hz, 1H), 4.66 (d, J = 13.5Hz, 1H), 5.24 (d, J = 13.5 Hz, 1H), 5.32 (s, 1H), 5.58 (s, 1H), 5.91 (d,J = 7.8 Hz, 1H), 6.81 (s, 2H), 7.06-7.20 (m, 5H). II-136

1H-NMR (CDCl3) δ: 1.26 (s, 3H), 1.33 (s, 3H), 2.96 (t, J = 11.9 Hz, 1H),3.46 (t, J = 10.6 Hz, 1H), 3.59 (t, J = 10.6 Hz, 1H), 3.77 (dd, J =11.9, 2.9 Hz, 1H), 3.95 (dd, J = 11.0, 2.9 Hz, 1H), 4.04-4.13 (m, 3H),4.56 (dd, J = 10.0, 2.9 Hz, 1H), 4.72 (d, J = 13.4 Hz, 1H), 5.27-5.31(m, 2H), 5.37 (s, 1H), 5.91 (d, J = 8.0 Hz, 1H), 6.87-6.91 (m, 2H),7.00-7.05 (m, 1H), 7.07-7.15 (m, 4H). II-137

1H-NMR (CDCl3) δ: 2.92 (t, J = 11.0 Hz, 1H), 3.38 (t, J = 11.0 Hz, 1H),3.56 (t, J = 10.4 Hz, 1H), 3.75 (d, J = 9.3 Hz, 1H), 3.81 (s, 3H), 3.95(d, J = 9.3 Hz, 1H), 4.06 (d, J = 13.9 Hz, 1H), 4.55 (d, J = 8.1 Hz,1H), 4.63 (d, J = 13.0 Hz, 1H), 5.27 (d, J = 13.9 Hz, 1H), 5.43 (br s,1H), 5.91 (d, J = 8.1 Hz, 1H), 6.09 (s, 1H), 6.82-6.86 (m, 1H), 6.93 (d,J = 8.1 Hz, 1H), 7.04-7.13 (m, 5H), 7.39-7.43 (m, 3H), 7.56-7.59 (m,2H). II-138

1H-NMR (CDCl3) δ: 2.94 (t, J = 11.3 Hz, 1H), 3.41 (t, J = 11.3 Hz, 1H),3.57 (t, J = 10.5 Hz, 1H), 3.76 (d, J = 11.0 Hz, 1H), 3.83 (s, 3H), 3.94(dd, J = 10.5, 2.7 Hz, 1H), 4.06 (d, J = 14.0 Hz, 1H), 4.55 (dd, J =9.5, 2.7 Hz, 1H), 4.68 (d, J = 12.6 Hz, 1H), 5.28 (d, J = 14.0 Hz, 1H),5.35 (s, 1H), 5.90 (d, J = 8.0 Hz, 1H), 6.05 (s, 1H), 6.84- 6.90 (m,2H), 7.00-7.15 (m, 5H), 7.38-7.42 (m, 3H), 7.56-7.60 (m, 2H).

TABLE 34 No. Structure NMR or LC/MS II-139

LC/MS (ESI): m/z = 614 [M + H]+, RT = 2.10 min, method (1) II-140

LC/MS (ESI): m/z = 614 [M + H]+, RT = 2.04 min, method (1) II-141

LC/MS (ESI): m/z = 614 [M + H]+, RT = 2.02 min, method (1) II-142

LC/MS (ESI): m/z = 670 [M + H]+, RT = 2.41 min, method (1)

TABLE 35 No. Structure NMR or LC/MS II-144

LC/MS (ESI): m/z = 575.20 [M + H]+, RT = 1.49 min, method (2) II-145

LC/MS (ESI): m/z = 575.00 [M + H]+, RT = 1.52 min, method (2) II-146

LC/MS (ESI): m/z = 657.90 [M + H]+, RT = 2.23 min, method (2)

The following compounds can be synthesized by the above methods as well.

Example 21: Method for Producing II-Form Crystals of Compound II-6

Acetonitrile (50 mL) and water (5 mL) were added to Compound II-6 (10.00g), the compound was heated to dissolve, and water (95 mL) was addedthereto. The solution was stirred for 10 minutes at room temperature,and the precipitated crystals were filtered. The obtained crystals weresubjected to through-flow drying to obtain II-form crystals (9.04 g) ofCompound II-6.

FIG. 4 shows powder X-ray diffraction results of II-form crystals ofCompound II-6.

Example 22: Method for Producing III-Form Crystals of Compound II-6

Methyl acetate (400 mL) was added to Compound II-6 (10.00 g), and thecompound was heated to dissolve. Methyl acetate (about 230 mL) of thesolution was concentrated under reduced pressure, the solution wasstirred for 70 minutes at room temperature, and the precipitatedcrystals were filtered. The obtained crystals were subjected tothrough-flow drying to obtain III-form crystals (7.87 g) of CompoundII-6.

FIG. 5 shows powder X-ray diffraction results of III-form crystals ofCompound II-6.

Biological test examples for compounds used in the present inventionwere described below.

Test Example 1: Measurement of Cap-Dependant Endonuclease (CEN)Inhibitory Activity

1) Preparation of Substrate

30merRNA(5′-pp-[m2′-O]GAA UAU(-Cy3) GCA UCA CUA GUA AGC UUU GCUCUA-BHQ2-3′: manufactured by Japan Bio Services Co., LTD.) in which G ata 5′ end is diphosphate-modified, a hydroxy group at 2′ position ismethoxylation-modified, U sixth from a 5′ end is labelled with Cy3, anda 3′ end is labelled with BHQ2 was purchased, and a cap structure wasadded using ScriptCap system manufactured by EPICENTRE (a product wasm7G [5′]-ppp-[5′] [m2′-O]GAA UAU(-Cy3) GCA UCA CUA GUA AGC UUU GCUCUA(BHQ2)-3′). This was separated and purified by denaturedpolyacrylamide gel electrophoresis, and used as a substrate.

2) Preparation of Enzyme

RNP was prepared from a virus particle using standard method (ReferenceDocument: VIROLOGY (1976) 73, p 327-338 OLGA M. ROCHOVANSKY).Specifically, A/WSN/33 virus (1×10³ PFU/mL, 200 μL) was inoculated in a10 days old embryonated chicken egg. After incubation at 37° C. for 2days, the allantoic fluid of the chicken egg was recovered. A virusparticle was purified by ultracentrifugation using 20% sucrose,solubilized using TritonX-100 and lysolecithin, and an RNP fraction(50-70% glycerol fraction) was collected by ultracentrifugation using a30-70% glycerol density gradient, and was used as an enzyme solution(containing approximately 1 nM PB1-PB2-PA complex).

3) Enzymatic Reaction

An enzymatic reaction solution (2.5 μL) (composition: 53 mMTris-hydrochloride (pH 7.8), 1 mM MgCl₂, 1.25 mM dithiothreitol, 80 mMNaCl, 12.5% glycerol, enzyme solution 0.15 μL) was dispensed into a384-well plate made of polypropylene. Then, 0.5 μL of a test compoundsolution which had been serially diluted with dimethyl sulfoxide (DMSO)was added to the plate. As a positive control (PC) or a negative control(NC), 0.5 μL of DMSO was added to the plate respectively. Each plate wasmixed well. Then, 2 μL of a substrate solution (1.4 nM substrate RNA,0.05% Tween20) was added to initiate a reaction. After room temperatureincubation for 60 minutes, 1 μL of the reaction solution was collectedand added to 10 μL of a Hi-Di formamide solution (containing GeneScan120 Liz Size Standard as a sizing marker: manufactured by AppliedBiosystems (ABI)) in order to stop the reaction. For NC, the reactionwas stopped in advance by adding EDTA (4.5 mM) before initiation of thereaction (all concentrations described above are final concentrations).

4) Measurement of Inhibition Ratio (IC₅₀ Value)

The solution for which the reaction was stopped was heated at 85° C. for5 minutes, rapidly cooled on ice for 2 minutes, and analyzed with an ABIPRIZM 3730 genetic analyzer. A peak of the cap-dependent endonucleaseproduct was quantitated by analysis software ABI Genemapper, a CENreaction inhibition ratio (%) of a test compound was obtained by settingfluorescent intensities of PC and NC to be 0% inhibition and 100%inhibition, respectively, an IC₅₀ value was obtained using curve fittingsoftware (XLfit2.0: Model 205 (manufactured by I DBS) etc.). The IC₅₀values of test substances being a parent compound, are shown in Table39.

Test Example 2: CPE Inhibitory Effect Confirming Assay

<Material>

2% FCS E-MEM (prepared by adding kanamycin and FCS to MEM (MinimumEssential Medium) (Invitrogen))

0.5% BSA E-MEM (prepared by adding kanamycin and BSA to MEM (MinimumEssential Medium) (Invitrogen))

HBSS (Hanks' Balanced Salt Solution)

MDBK cell

Cells were adjusted to the appropriate cell number (3×10⁵/mL) with 2%FCS E-MEM.

MDCK cell

After washing with HBSS two times, cells were adjusted to theappropriate cell number (5×10⁵/mL) with 0.5% BSA E-MEM.

Trypsin solution

Trypsin from porcine pancreas (SIGMA) was dissolved in PBS(−), andfiltrated with a 0.45 μm filter.

EnVision (PerkinElmer)

WST-8 Kit (Kishida Chemical Co., Ltd.)

10% SDS solution

<Operation Procedure>

Dilution and Dispensation of Test Sample

As a culture medium, 2% FCS E-MEM was used at the use of MDBK cells, and0.5% BSA E-MEM was used at the use of MDCK cells. Hereinafter, fordiluting virus, cells and a test sample, the same culture medium wasused.

A test sample was diluted with a culture medium to an appropriateconcentration in advance, and then 2 to 5-fold serial dilution on a 96well plate (50 μL/well) was prepared. Two plates, one for measuringanti-Flu activity and the another for measuring cytotoxity, wereprepared. Each assay was performed triplicate for each drug.

At the use of MDCK cells, Trypsin was added to the cells to be a finalconcentration of 3 μg/mL only for measuring anti-Flu activity.

Dilution and Dispensation of Influenza Virus

An influenza virus was diluted with a culture medium to an appropriateconcentration in advance, and each 50 μL/well was dispensed on a 96-wellplate containing a test substance. Each 50 μL/well of a culture mediumwas dispensed on a plate containing a test substance for measuringcytotoxity.

Dilution and Dispensation of Cell

Each 100 μL/well of cells which had been adjusted to the appropriatecell number was dispensed on a 96 well plate containing a test sample.

This was mixed with a plate mixer, and incubated in a CO2 incubator for3 days for measuring anti-Flu activity and measuring cytotoxity.

Dispensation of WST-8

The cells in the 96-well plate which had been incubated for 3 days wasobserved visually under a microscope, and appearance of the cells, thepresence or absence of a crystal of test substance were checked. Thesupernatant was removed so that the cells were not absorbed from theplate.

WST-8 Kit was diluted 10-fold with a culture medium, and each 100 μL wasdispensed into each well. After mixing with a plate mixer, cells wereincubated in a CO2 incubator for 1 to 3 hours.

After incubation, regarding the plate for measuring anti-Flu activity,each 10 μL/well of a 10% SDS solution was dispensed in order toinactivate a virus.

Measurement of Absorbance

After the 96-well plate was mixed, absorbance was measured with EnVisionat two wavelengths of 450 nm/620 nm.

<Calculation of Each Measurement Item Value>

The value was calculated using Microsoft Excel or a program having theequivalent calculation and processing ability, based on the followingcalculation equation.

Calculation of effective inhibition concentration to achieve 50%influenza infected cell death (EC50)EC50=10^(Z)Z=(50%−High %)/(High %−Low %)×{log(High conc.)−log(Low conc.)}+log(Highconc.)

For the parent compounds used in the present invention, measurementresults of Test Example 1 and Test Example 2 are shown below.

TABLE 36 CEN_IC50 CPE_EC50 No. nM nM III-2 1.93 1.13 III-3 2.22 3.39III-9 2.17 10.90 III-10 4.05 3.46 III-11 13.10 9.98 III-12 2.18 3.38III-13 3.94 4.00 III-14 15.00 15.70 III-19 2.37 1.43 III-20 3.24 4.00III-21 4.06 2.70 III-22 3.46 3.07 III-23 1.48 0.86 III-26 1.63 3.00III-28 10.70 5.67 III-29 0.87 0.66 III-30 5.68 3.01 III-31 18.50 3.17III-33 2.08 2.36 III-34 4.69 2.85 III-35 3.86 3.00 III-36 2.37 2.45III-37 4.24 3.43 III-38 8.26 4.04 III-39 2.75 2.81 III-40 2.99 2.95III-41 2.10 2.17 III-42 3.93 2.64 III-43 3.90 3.18 III-44 3.81 3.68III-45 1.63 3.07 III-46 2.91 3.18 III-47 2.25 2.53 III-48 3.49 3.57III-49 6.79 4.17 III-50 2.55 4.36 III-51 2.22 2.58 III-52 3.62 3.28

TABLE 37 CEN CPE IC50 IC50 No. nM nM III-53 2.46 3 III-54 1.27 1.18III-55 2.13 3.45 III-57 4.27 3.47 III-58 2.65 3.13 III-59 0.57 3.11

Based on the above results, the parent compounds used in the presentinvention exhibit high cap-dependent endonuclease (CEN) inhibitoryactivity and/or high CPE inhibitory effect and thus can be a usefulagent for treatment and/or prevention of symptom and/or disease inducedby infection with influenza virus.

Test Example 3: CYP Inhibition Test

Using commercially available pooled human hepatic microsome, andemploying, as markers, 7-ethoxyresorufin O-deethylation (CYP1A2),tolbutamide methyl-hydroxylation (CYP2C9), mephenytoin 4′-hydroxylation(CYP2C19), dextromethorphan O-demethylation (CYP2D6), and terfenedinehydroxylation (CYP3A4) as typical substrate metabolism reactions ofhuman main five CYP enzyme forms (CYP1A2, 2C9, 2C19, 2D6, 3A4), aninhibitory degree of each metabolite production amount by a compoundused in the present invention was assessed.

The reaction conditions were as follows: substrate, 0.5 μmol/Lethoxyresorufin (CYP1A2), 100 μmol/L tolbutamide (CYP2C9), 50 μmol/LS-mephenytoin (CYP2C19), 5 μmol/L dextromethorphan (CYP2DG), 1 μmol/Lterfenedine (CYP3A4); reaction time, 15 minutes; reaction temperature,37° C.; enzyme, pooled human hepatic microsome 0.2 mg protein/mL;concentration of a compound used in the present invention, 1, 5, 10, 20μmol/L (four points).

Each five kinds of substrates, human hepatic microsome, or a compoundused in the present invention in 50 mmol/L Hepes buffer as a reactionsolution was added to a 96-well plate at the composition as describedabove, NADPH, as a cofactor was added to initiate metabolism reactionsas markers and, after the incubation at 37° C. for 15 minutes, amethanol/acetonitrile=1/1 (v/v) solution was added to stop the reaction.After the centrifugation at 3000 rpm for 15 minutes, resorufin (CYP1A2metabolite) in the supernatant was quantified by a fluorescentmultilabel counter and toltributamide hydroxide (CYP2C9P metabolite),mephenytoin 4′ hydroxide (CYP2C19 metabolite), dextromethorphan (CYP2D6metabolite), and terfenadine alcohol (CYP3A4 metabolite) were quantifiedby LC/MS/MS.

Addition of only DMSO being a solvent dissolving a compound used in thepresent invention to a reaction system was adopted as a control (100%),remaining activity (%) was calculated at each concentration of acompound used in the present invention added as the solution and IC50was calculated by reverse presumption by a logistic model using aconcentration and an inhibition rate.

(Result)

Compound III-2: five kinds >20 μmol/L

Test Example 4: BA Test

Materials and methods for experiments to evaluate oral absorption

(1) Experimental animals: mice or SD rats were used.

(2) Rearing condition: mice or SD rats were allowed free access to solidfeed and sterilized tap water.

(3) Setting of dosage and grouping: Oral administration and intravenousadministration were performed with the predetermined dosage. Groupingwas set as below. (Dosage was changed per compound)

Oral administration 1 to 30 mg/kg (n=2 to 3)

Intravenous administration 0.5 to 10 mg/kg (n=2 to 3)

(4) Preparation of administration solutions: Oral administration wasperformed as solution or suspension. Intravenous administration wasperformed after solubilization.

(5) Routes of administration: Oral administration was performedmandatory into the stomach by oral sonde. Intravenous administration wasperformed from caudal vein by syringes with needle.

(6) Evaluation items: Blood was collected serially and concentration ofa compound used in the present invention in plasma was measured byLC/MS/MS.

(7) Statistical analysis: About transition of concentration of acompound used in the present invention in plasma, the area under theplasma concentration versus time curve (AUC) was calculated bynon-linear least-squares method program, WinNonlin (a registeredtrademark), and bioavailability (BA) of a compound used in the presentinvention was calculated from AUCs of the oral administration group andthe intravenous administration group.(Result)Compound II-6: 14.9%Compound III-2: 4.2%

Based on the above results, the prodrug had improved bioavailabilityother than the parent compound.

Therefore, the compound used in the present invention has excellent oralabsorbability and can be a useful agent for treatment and/or preventionof symptom and/or disease induced by infection with influenza virus.

Test Example 5: Metabolism Stability Test

Using commercially available pooled human hepatic microsomes, a compoundused in the present invention was reacted for a constant time, and aremaining rate was calculated by comparing a reacted sample and anunreacted sample, thereby, a degree of metabolism in liver was assessed.

A reaction was performed (oxidative reaction) at 37° C. for 0 minute or30 minutes in the presence of 1 mmol/L NADPH in 0.2 mL of a buffer (50mmol/L Tris-HCl pH 7.4, 150 mmol/L potassium chloride, 10 mmol/Lmagnesium chloride) containing 0.5 mg protein/mL of human livermicrosomes. After the reaction, 50 μL of the reaction solution was addedto 100 μL of a methanol/acetonitrile=1/1 (v/v), mixed and centrifuged at3000 rpm for 15 minutes. The compound used in the present invention inthe supernatant was quantified by LC/MS/MS or Solid Phase Extraction(SPE)/MS, and a remaining amount of the compound used in the presentinvention after the reaction was calculated, letting a compound amountat 0 minute reaction time to be 100%. Hydrolysis reaction was performedin the absence of NADPH and glucuronidation reaction was in the presenceof 5 mM UDP-glucuronic acid in place of NADPH, followed by similaroperations.

(Result) % inhibition was shown at 2 μmol/L of test compound.

Compound III-2: 90.1%

Test Example 6: CYP3A4 Fluorescent MBI Test

The CYP3A4 fluorescent MBI test is a test of investigating enhancementof CYP3A4 inhibition of a compound used in the present invention by ametabolism reaction, and the test was performed using, as CYP3A4 enzymeexpressed in Escherichia coli and employing, as an index, a reaction inwhich 7-benzyloxytrifluoromethylcoumarin (7-BFC) is debenzylated by theCYP3A4 enzyme to produce a metabolite, 7-hydroxytrifluoromethylcoumarin(HFC) emitting fluorescent light.

The reaction conditions were as follows: substrate, 5.6 μmol/L 7-BFC;pre-reaction time, 0 or 30 minutest reaction time, 15 minutest reactiontemperature, 25° C. (room temperature); CYP3A4 content (expressed inEscherichia coli), at pre-reaction 62.5 μmol/mL, at reaction 6.25μmol/mL (at 10-fold dilution); test drug concentration of a compoundused in the present invention, 0.625, 1.25, 2.5, 5, 10, 20 μmol/L (sixpoints).

An enzyme in a K-Pi buffer (pH 7.4) and a solution of a compound used inthe present invention as a pre-reaction solution were added to a 96-wellplate at the above composition of the pre-reaction, a part of it wastransferred to another 96-well plate so that it was 1/10 diluted with asubstrate and a K-Pi buffer, NADPH as a co-factor was added to initiatea reaction as an index (without preincubation) and, after apredetermined time of a reaction, acetonitrile/0.5 mol/L Tris(trishydroxyaminomethane)=4/1 (V/V) was added to stop the reaction. Inaddition, NADPH was added to a remaining preincubation solution toinitiate a preincubation (with preincubation) and, after a predeterminedtime of a preincubation, a part was transferred to another plate so thatit was 1/10 diluted with a substrate and a K-Pi buffer to initiate areaction as an index. After a predetermined time of a reaction,acetonitrile/0.5 mol/L Tris (trishydroxyaminomethane)=4/1 (V/V) wasadded to stop the reaction. For the plate on which each index reactionhad been performed, a fluorescent value of 7-HFC which is a metabolitewas measured with a fluorescent plate reader. (Ex=420 nm, Em=535 nm).

Addition of only DMSO which is a solvent dissolving a compound used inthe present invention to a reaction system was adopted as a control(100%), remaining activity (%) was calculated at each concentration of acompound used in the present invention added as the solution, and IC₅₀was calculated by reverse-presumption by a logistic model using aconcentration and an inhibition rate. When a difference between IC₅₀values is 5 μmol/L or more, this was defined as (+) and, when thedifference is 3 μmol/L or less, this was defined as (−).

(Result)

Compound III-2: (−)

Test Example 7: Fluctuation Ames Test

Mutagenicity of compounds used in the present invention was evaluated.

20 μL of freezing-stored rat typhoid bacillus (Salmonella typhimuriumTA98 strain, TA100 strain) was inoculated on 10 mL of a liquid nutrientmedium (2.5% Oxoid nutrient broth No. 2), and this was cultured beforeshaking at 37° C. for 10 hours. 9 mL of a bacterial solution of the TA98strain was centrifuged (2000×g, 10 minutes) to remove a culturingsolution. The bacteria was suspended in 9 mL of a Micro F buffer(K₂HPO₄: 3.5 g/L, KH₂PO₄: 1 g/L, (NH₄)₂SO₄: 1 g/L, trisodium citratedehydrate: 0.25 g/L, MgSO₄.7H₂O: 0.1 g/L), the suspension was added to110 mL of an Exposure medium (Micro F buffer containing Biotin: 8 μg/mL,histidine: 0.2 μg/mL, glucose: 8 mg/mL). The TA100 strain was added to120 mL of the Exposure medium relative to 3.16 mL of the bacterialsolution to prepare a test bacterial solution. Each 12 μL of DMSOsolution of a compound used in the present invention (several stagedilution from maximum dose 50 mg/mL at 2 to 3 fold ratio), DMSO as anegative control, and 50 μg/mL of 4-nitroquinoline-1-oxide DMSO solutionfor the TA98 strain, 0.25 μg/mL of2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide DMSO solution for the TA100strain under the non-metabolism activating condition, 40 μg/mL of2-aminoanthracene DMSO solution for the TA98 strain, 20 μg/mL of2-aminoanthracene DMSO solution for the TA100 strain under themetabolism activating condition as a positive control, and 588 μL of thetest bacterial solution (a mixed solution of 498 μl of the testbacterial solution and 90 μL of S9 mix under the metabolism activatingcondition) were mixed, and this was shaking-cultured at 37° C. for 90minutes. 460 μL of the bacterial solution exposed to a compound used inthe present invention was mixed with 2300 μL of an Indicator medium(Micro F buffer containing biotin: 8 μg/mL, histidine: 0.2 μg/mL,glucose: 8 mg/mL, Bromo Cresol Purple: 37.5 μg/mL), each 50 μL wasdispensed into microplate 48 wells/dose, and this was subjected tostationary culturing at 37° C. for 3 days. Since a well containing abacterium which has obtained the proliferation ability by mutation of anamino acid (histidine) synthesizing enzyme gene turns from purple toyellow due to a pH change, the bacterium proliferation well which hasturned to yellow in 48 wells per dose is counted, and was assessed bycomparing with a negative control group. (−) means that mutagenicity isnegative and (+) is positive.

(Result)

Compound III-2: (−)

Test Example 8: hERG Test

For the purpose of assessing risk of an electrocardiogram QT intervalprolongation of the compound used in the present invention, effects ofthe compound used in the present invention on delayed rectifier K+current (IKF), which plays an important role in the ventricularrepolarization process, was studied using HEK293 cells expressing humanether-a-go-go related gene (hERG) channel.

After a cell was retained at a membrane potential of −80 mV by wholecell patch clamp method using an automated patch clamp system(PatchXpress 7000A, Axon Instruments Inc.), I_(Kr) induced bydepolarization pulse stimulation at +40 mV for 2 seconds and, further,repolarization pulse stimulation at −50 mV for 2 seconds, was recorded.After the generated current was stabilized, extracellular solution(NaCl: 135 mmol/L, KCl: 5.4 mmol/L, NaH₂PO₄: 0.3 mmol/L, CaCl₂.2H₂O: 1.8mmol/L, MgCl₂.6H₂O: 1 mmol/L, glucose: 10 mmol/L, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid): 10 mmol/L,pH=7.4), in which the compound used in the present invention had beendissolved at an objective concentration, was applied to the cell at roomtemperature for 10 minutes. From the recording I_(Kr), an absolute valueof the tail peak current was measured based on the current value at theresting membrane potential using analysis software (DataXpress ver. 1,Molecular Devices Corporation). Further, the % inhibition relative tothe tail peak current before application of the compound used in thepresent invention was calculated, and compared with the vehicle-appliedgroup (0.1% dimethyl sulfoxide solution) to assess influence of thecompound used in the present invention on I Hr.

(Result) % inhibition was shown al 0.3 to 10 μM of test compound.

Compound III-2: 7.9%

Test Example 9: Solubility Test

The solubility of the compound used in the present invention wasdetermined under 1% DMSO addition conditions. A 10 mmol/L solution ofthe compound was prepared with DMSO, and 2 μL of the solution of thecompound used in the present invention was added, respectively, to 198μL of JP-1 solution (water were added to 2.0 g of sodium chloride and7.0 mL of hydrochloric acid to reach 1000 mL) and JP-2 solution (1volume of water were added to 1 volume of the solution which 3.40 g ofpotassium dihydrogen phosphate and 3.55 g of anhydrous disodium hydrogenphosphate to reach 1000 mL). The mixture was shaken for 1 hour at a roomtemperature, and the mixture was filtered. The filtrate was ten-folddiluted with methanol/water=1/1 (v/v), and the compound concentration inthe filtrate was measured with LC/MS or SPE/MS by the absolutecalibration method.

(Result)

Compound III-2: 42.2 μmol/L

Test Example 10: Powder Solubility Test

Appropriate amounts of the compound used in the present invention wasput into vials and 200 μL of JP-1st Fluid (water was added to 2.0 g ofsodium chloride in 7.0 mL of hydrochloride acid to reach 1000 mL),JP-2nd Fluid (water was added to 500 mL of phosphate buffer solutionwith a pH of 6.8) and 20 mmol/L sodium taurocholate (TCA)/JP-2nd Fluid(JP-2nd Fluid was added to 1.08 g of TCA in JP-2nd Fluid to reach 100mL) was added to each vial. When the compound was completely dissolved,appropriate amount of compound was added. After shaken for 1 hour at 37°C., the mixture was filtered and 100 μL of methanol was added to 100 μLof each filtrate (double dilution). Dilution magnification was changedif necessary. After it was confirmed whether there were air bubbles andprecipitates in the vials, the vials were shaken with tight stopper. Thecompound concentration was determined with HPLC by the absolutecalibration method.

(Result)

Compound III-2: JP-1 solution; 7.1 μg/mL, JP-2 solution; 4.4 μg/mL, 20mmol/L TCA/JP-2 solution; 16.1 μg/mL

Test Example 11: Ames Test

Ames test was performed by using Salmonellas (Salmonella typhimurium) TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA as teststrains with or without metabolic activation in the pre-incubationmethod to check the presence or absence of gene mutagenicity ofcompounds used in the present invention.

(Result)

Compound III-2: (−)

Test Example 12: Light Hemolysis Test

The compound used in the present invention was dissolved at targetconcentrations and was mixed with a 2.5 v/v % suspension of red bloodcells prepared from a defibrinated blood of sheep on a microplate atconcentrations of 0.0008 to 0.1 w/v %. The mixtures were exposed to 10°J/cm² of UV-irradiation within a range of wavelength 290 to 400 nm, UVAand UVB using ultra violet fluorescent lamps, GL20SE and FL20S-BLB lampsmanufactured by Sankyo Denki Co., Ltd. and Panasonic Corporation,respectively. After the completion of the irradiation, the mixtures werecentrifuged, and a supernatant of the mixture was collected and waslocated on a microplate. The phototoxicity was assessed by measuring anabsorbance at wavelength of 540 nm and 630 nm in the supernatant. Theabsorbance data at wavelength of 540 nm and 630 nm were used asindicators of biomembrane damage (photohemolysis %) and hyperoxidationof lipid membrane (methemoglobin formation), respectively. The criteriaof phototoxicity was as follows; It was judged to be non-phototoxic (−)when the photohemolysis %<10 and the maximal change in the absorbance at630 nm (ΔOD)<0.05 were observed. It was judged to be non-phototoxic (+)when the photohemolysis was more than 10% and the maximal change in theabsorbance at 630 nm (ΔOD) was more than 0.05.

(Result)

Compound III-2: (−)

Test Example 13: Transition of Plasma Concentration

The plasma concentration of Compound III-2 and Compound II-6 after oraladministration of prodrug Compound II-6, the parent compound of whichwas Compound III-2, to rat under non-fasting conditions was measured.The result is shown in FIGS. 1 and 2.

The concentration of Compound II-6 in all plasma samples was adetermination limit or less. Therefore, prodrug Compound II-6, theparent compound of which was Compound III-2 was found to have changedpromptly to Compound III-2 in vivo after administration (see FIG. 2).

Based on the above test results, it was revealed that the compoundconverted into a prodrug was absorbed into the body after oraladministration, and rapidly converted into a parent compound in theblood. Therefore, the compounds (parent compounds and/or prodrugs) usedin the present invention can be useful agents for treatment and/orprevention of symptoms and/or diseases induced by infection withinfluenza virus.

Test Example 14: Intravenous Administration Test

Examined experimental materials and method of intravenous administrationtest

(1) Animals used: SD rats were used.

(2) Rearing conditions: Pellets and sterilized tap water were fed to SDrats ad libitum.

(3) Dosage and grouping: A predetermined dosage was intravenouslyadministered. Groups were set as follows. (Dosage varied for eachcompound)

Intravenous administration 0.5-1 mg/kg (n=2-3)

(4) Preparation of administration solution: Intravenous administrationwas performed after solubilization.

(5) Administration method: Intravenous administration was performed witha needle-equipped syringe on the caudal vein.

(6) End point: Blood was collected over time, and the plasmaconcentration of the compound used in the present invention was measuredusing LC/MS/MS.

(7) Statistical analysis: As for the transition of the plasmaconcentration of the compound used in the present invention, the totalbody clearance (CLtot) and the elimination half-life (t½, z) werecalculated using nonlinear least-squares program WinNonlin (Registeredtrademark).(Results)Compound No. III-2:CLtot: 16.4 mL/min/kgt½, z: 3.4 hours

From the above results, it was found that Compound III-2 is a compoundhaving a low total body clearance and a long half-life.

Therefore, the compound used in the present invention has excellentpersistence and can be a useful agent for treatment and/or prevention ofsymptom and/or disease induced by infection with influenza virus.

Test Example 15: Clinical Test

The efficacy and safety of a single oral administration of aninvestigational drug (active ingredient (Compound II-6): 10 mg, 20 mg,40 mg) to patients infected by influenza virus were evaluated by arandomized, placebo-controlled, double-blind comparative study. As forthe primary endpoint, subjects made evaluations by themselves on a4-point scale [0: none, 1: mild, 2: moderate, 3: severe] concerning thetime to alleviation of influenza symptoms (the time from the beginningof administration of the investigational drug until 7 influenza symptoms(“cough”, “sore throat”, “headache”, “nasal congestion”, “feverishnessor chills”, “muscular or joint pain”, and “fatigue”) were alleviated) toevaluate the efficacy of the investigational drug over the placebo.

Patients who satisfied all of the following criteria were selected assubjects.

(a) Male or female patients at 20 years old or older and younger than 65years old

(b) Patients satisfying all of the following criteria and diagnosed withinfluenza virus infectious disease

Positive in influenza rapid diagnosis [Rapid antigen test (RAT)] basedon a nasal or throat swab

Body temperature (axillary temperature) of 38.0° C. or higher

Having one or more moderate or severer symptoms among the followingsystemic symptoms and respiratory symptoms due to influenza virusinfectious disease

-   -   Systemic symptoms (headache, feverishness or chills, muscular or        joint pain, fatigue)    -   Respiratory symptoms (cough, sore throat, nasal congestion)        (C) Patients within 48 hours from onset (at registration)        The definition of onset is any of the following.

When the body temperature increased for the first time (at least anincrease of 1° C. from normal temperature)

When any one or more of the systemic symptoms and respiratory symptomswere developed

Method for Administering Investigational Drug

(i) Test Drug

10 mg Tablet of Compound II-6: White to pale yellowish white, circular,film-coated tablet containing 10 mg of Compound II-6

20 mg Tablet of Compound II-6: White to pale yellowish white,elliptical, film-coated tablet containing 20 mg of Compound II-6

(ii) Placebo or Control Drug

Placebo for 10 mg tablet of Compound II-6: Tablet undistinguishable from10 mg tablet of Compound II-6

Placebo for 20 mg tablet of Compound II-6: Tablet undistinguishable from20 mg tablet of Compound II-G

Dosage and Administration Method

Eligible subjects were randomly allocated to a Compound II-6 10 mggroup, 20 mg group, 40 mg group, and placebo group in a ratio of1:1:1:1. Subjects received a single oral administration of total 3tables of Compound II-6 tablets and/or placebo tablets in a combinationindicated in the following table on Day 1.

Investigational Drug for Each Administered Group

TABLE 38 Compound Compound Placebo tablet Placebo tablet Treatment II-6II-6 Matching Compound Matching Compound Groups 10 mg tablet 20 mgtablet II-6 10 mg tablet II-6 20 mg tablet Compound II-6 1 tablet — — 2tablets 10 mg tablet Compound II-6 — 1 tablet 1 tablet 1 tablet 20 mgtablet Compound II-6 — 2 tablets 1 tablet — 40 mg tablet Placebo — — 1tablet 2 tabletsMain Efficacy Endpoint

The main efficacy endpoint is the time to alleviation of influenzasymptoms (the time to alleviation of influenza symptoms).

It is the time from the beginning of administration until alleviation ofinfluenza symptoms. Alleviation of influenza symptoms refers to when all7 influenza symptoms (cough, sore throat, headache, nasal congestion,feverishness or chills, muscular or joint pain, fatigue) become “0:none” or “1: mild” in the patient diary that the subject keeps, and thiscondition continues at least 21.5 hours (24 hours—10%).

Secondary Efficacy Endpoint

The secondary efficacy endpoint is as follows.

(1) Time to Alleviation of Each Influenza Symptom

It is the time from the beginning of administration until alleviation ofeach influenza symptom. Alleviation of a symptom refers to when thetarget item becomes “0: none” or “1: mild”, and this condition continuesat least 21.5 hours (24 hours—10%).

Analysis of Primary Endpoint

As for the time to alleviation of influenza symptoms, which is theprimary endpoint, the primary analysis and the secondary analysis aredescribed. In addition to the ITTI group, the primary analysis was alsoperformed on the PPS group for sensitivity analysis. Other analyses wereperformed only on the ITTI group.

(1) Primary Analysis

The hazard ratio, 95% confidence interval, and P value of eachadministered group relative to the placebo group were calculated by aCox proportional hazard model using the time to alleviation of influenzasymptoms as a response, the administered groups as fixed effects, andthe current smoking habit and the total score of 7 influenza symptoms albaseline before administration, which are allocation factors, ascovariates. In order to prevent an increase of the probability of type Ierror due to performing the test multiple times, the P value wasadjusted by the Hommel's method.

(2) Secondary Analysis

The placebo group and each investigational drug administered group werecompared by stratified generalized Wilcoxon test using the time toalleviation of influenza symptoms as a response, the administered groupsas explanatory variables, and the category (11 points or less, 12 pointsor more) of the total score of 7 influenza symptoms beforeadministration and the smoking habit, which are allocation factors, asstratification factors.

Also, a Kaplan-Meier survival curve was drawn for each group tocalculate the median time to alleviation of influenza symptoms and the95% confidence interval thereof. The Greenwood's method was used forcalculating the confidence interval.

Analysis of Secondary Endpoint

(1) Time Until Each Alleviation of Influenza Symptom

The same analysis as in the primary endpoint was performed, with thetime until each alleviation of influenza symptom being regarded as aresponse. At this time, cases where the symptom before administrationwas “0: none” or “1: mild” were excluded from the analysis target.

(1) Results of Primary Endpoint (Time to Alleviation of InfluenzaSymptoms)

Out of 400 randomly selected patients, 389 patients (98 patients (98%)in the 10 mg administered group, 95 patients (95%) in the 20 mgadministered group, 99 patients (99%) in the 40 mg administered group,and 97 patients (97%) in the placebo group) completed the test. As forthe primary endpoint, the ITTI Population (cases where aninvestigational drug was administered, and influenza virus infection wasconfirmed) consisted of 400 patients.

The per protocol set cases consisted of 368 patients (89 patients (89%)in the 10 mg administered group, 92 patients (92%) in the 20 mgadministered group, 96 patients (96%) in the 40 mg administered group,and 91 patients (91%) in the placebo group). As for the ITTI Populationof each group, it was found from the rapid antigen detection test that75% to 79% of the patients were infected by influenza A virus, and 21%to 25% of the patients were infected by influenza B virus.

Analysis results are shown in the following tables.

TABLE 39 Testdrug Testdrug Testdrug 10 mg 20 mg 40 mg Placeboadministered group administered group administered group administeredgroup N (human) 100 100 100 100 Median value 54.2 51 49.5 77.7 (hour)95% Confidence 47.7, 66.8 44.5, 62.4 44.5, 64.4 67.6, 88.7 interval(hour) Difference from −23.4 −26.6 −28.2 — placebo (hour) GeneratedWilson test P value 0.0085 0.0182 0.0046 — Cox proportional hazard modelrelative to placebo Hazard ratio 0.758 0.81 0.817 — 95% Confdence 0.571,1.007 0.608, 1.078 0.614, 1.087 — interval P value 0.0561 0.1488 0.165 —

The primary endpoint of this test, i.e., the median time until thesymptoms were alleviated, was 54.2 hours in the 10 mg administered group(95% CI: 47.7, 66.8), 51.0 hours in the 20 mg administered group (95%CI: 47.7, 66.8), 49.5 hours in the 40 mg administered group (95% CI:44.5, 64.4), and 77.7 hours in the placebo group (95% CI: 67.6, 88.7).

(2) Time Until Each of the Seven Symptoms is Alleviated

The following tables show the results of analyzing the time until eachof the 7 influenza symptoms (“cough”, “sore throat”, “headache”, “nasalcongestion”, “feverishness or chills”, “muscular or joint pain”,“fatigue”) is alleviated.

(i) Time Until “Nasal Congestion” Symptom is Alleviated

TABLE 40 Testdrug Testdrug Testdrug 10 mg 20 mg 40 mg Placeboadministered group administered group administered group administeredgroup N (human) 49 38 45 47 Median value 25.2 21.6 21.9 42.8 (95% CI)(hour) (19.0, 47.2) (13.4, 30.5) (16.0, 28.7) (22.9, 68.3) Differencefrom −17.6 −21.3 −21 — placebo (hour) P value (G. 0.043 0.0516 0.0003 —Wilcoxon test)^(a) Hazard ratio 0.742 0.59 0.564 — (95% CI)^(b) (0.494,1.114) (0.379, 0.920) (0.369, 0.862) P value 0.15 0.0199 0.0081 — (Coxmodel)^(b)(ii) Time Until “Muscular or Joint Pain” Symptom is Alleviated

TABLE 41 Testdrug Testdrug Testdrug 10 mg 20 mg 40 mg Placeboadministered group administered group administered group administeredgroup N (human) 73 77 71 71 Median value 31.2 29.9 25.4 41.9 (95% CI)(hour) (24.9, 39.9) (22.8, 37.0) (20.5, 28.9) (28.7, 48.6) Differencefrom −10.7 −12 −16.4 — placebo (hour) P value (G. 0.2153 0.0346 0.0048 —Wilcoxon test)^(a) Hazard ratio 0.77 0.687 0.657 — (95% CI)^(b) (0.553,1.072) (0.494, 0.955) (0.469, 0.920) P value 0.1217 0.0255 0.0145 — (Coxmodel)^(b)(iii) Time Until “Fatigue” Symptom is Alleviated

TABLE 42 Testdrug Testdrug Testdrug 10 mg 20 mg 40 mg Placeboadministered group administered group administered group administeredgroup N (human) 82 82 77 79 Median value 32 31.3 31.1 42.7 (95% CI)(hour) (29.2, 39.9) (26.7, 42.4) (24.6, 38.6) (30.3, 53.2) Differencefrom −10.7 −11.5 −11.7 — placebo (hour) P value (G. 0.1221 0.0594 0.0224— Wilcoxon test)^(a) Hazard ratio 0.783 0.876 0.724 — (95% CI)^(b)(0.574, 1.069) (0.637, 1.203) (0.527, 0.995) P value 0.1236 0.412 0.0463— (Cox model)^(b)(iv) Time Until “Feverishness or Chills” Symptom is Alleviated

TABLE 43 Testdrug Testdrug Testdrug 10 mg 20 mg 40 mg Placeboadministered group administered group administered group administeredgroup N (human) 97 93 94 95 Median value 24.7 29.4 23 28.8 (95% CI)(hour) (21.3, 28.4) (22.0, 34.8) (19.8, 28.6) (21.1, 33.4) Differencefrom −4.1 0.6 −5.8 — placebo (hour) P value (G. 0.0602 0.3774 0.0258 —Wilcoxon test)^(a) Hazard ratio 0.635 0.848 0.71 — (95% CI)^(b) (0.475,0.850) (0.6347, 1.133) (0.529, 0.951) P value 0.0023 0.2642 0.0216 —(Cox model)^(b)(v) Time Until “Headache” Symptom is Alleviated

TABLE 44 Testdrug Testdrug Testdrug 10 mg 20 mg 40 mg Placeboadministered group administered group administered group administeredgroup N (human) 61 58 54 57 Median value 42.2 37 37.9 43.7 (95% CI)(hour) (29.8, 47.3) (28.5, 43.5) (28.6, 44.5) (29.7, 53.6) Differencefrom −1.5 −6.7 −5.8 — placebo (hour) P value (G. 0.6846 0.7741 0.0904 —Wilcoxon test)^(a) Hazard ratio 0.803 0.936 0.655 — (95% CI)^(b) (0.557,1.157) (0.635, 1.381) (0.447, 0.961) P value 0.2388 0.7404 0.0304 — (Coxmodel)^(b)(vi) Time Until “Cough” Symptom is Alleviated

TABLE 45 Testdrug Testdrug Testdrug 10 mg 20 mg 40 mg Placeboadministered group administered group administered group administeredgroup N (human) 74 74 78 75 Median value 31.1 29.8 24.6 31.2 (95% CI)(hour) (21.3, 41.5) (21.9, 32.9) (16.1, 29.4) (20.9, 51.4) Differencefrom −0.1 −1.4 −6.6 — placebo (hour) P value (G. 0.6643 0.8536 0.1551 —Wilcoxon test)^(a) Hazard ratio 0.941 0.883 0.865 — (95% CI)^(b) (0.675,1.312) (0.636, 1.226) (0.626, 1.196) P value 0.7188 0.4569 0.3796 — (Coxmodel)^(b)(vii) Time Until “Sore Throat” Symptom is Alleviated

TABLE 46 Testdrug Testdrug Testdrug 10 mg 20 mg 40 mg Placeboadministered group administered group administered group administeredgroup N (human) 56 64 55 46 Median value 35.3 27.8 31.9 26.3 (95% CI)(hour) (21.2, 49.8) (19.9, 32.1) (17.3, 43.0) (16.5, 45.2) Differencefrom 9.1 1.5 5.6 — placebo (hour) P value (G. 0.2905 0.6293 0.993 —Wilcoxon test)^(a) Hazard ratio 1.312 1.05 1.092 — (95% CI)^(b) (0.882,1.951) (0.713, 1.547) (0.738, 1.617) P value 0.18 0.8047 0.6602 — (Coxmodel)^(b)a Stratified generalized Wilson test relative to placebo. Stratificationfactors: Smoking habit, and composite symptom scores at baseline.b Cox proportional hazard model relative to placebo. Covariates: Smokinghabit, and composite symptom scores at baseline.Subset of patients whose score of symptoms at baseline was “moderate” or“severe” CI: Confidence interval

An analysis using a Cox proportional hazard model revealed that the 40mg administered group in comparison to the placebo group showed asignificant decrease in time until the following 5 symptoms: “nasalcongestion”, “muscular or joint pain”, “fatigue”, “feverishness orchills”, and “headache” were alleviated. For example, as for 2 symptoms,i.e., “nasal congestion” and “muscular or joint pain”, the median timesuntil these symptoms were improved were 21.0 hours and 16.4 hours,respectively, and they were shorter in the 40 mg administered group thanthe placebo group.

Statistically significant differences were observed also in the 10 mgadministered group and the 20 mg administered group with respect to thefollowing symptoms: “muscular or joint pain”, “nasal congestion”, and“feverishness or chills”.

Test Example 16: Clinical Test (Ph3: Adults and Adolescents)

The efficacy and safety of a single oral administration of aninvestigational drug (active ingredient (Compound II-6): 40 mg, 80 mg)to patients infected by influenza virus were evaluated by a randomized,double-blind comparative study in comparison to 75 mg Oseltamiviradministered twice per day for 5 days or a placebo. As for the primaryendpoint, subjects made evaluations by themselves on a 4-point scale [0:none, 1: mild, 2: moderate, 3: severe] concerning the time toalleviation of influenza symptoms (the time from the beginning ofadministration of the investigational drug until 7 influenza symptoms(“cough”, “sore throat”, “headache”, “nasal congestion”, “feverishnessor chills”, “muscular or joint pain”, and “fatigue”) were alleviated) toevaluate the efficacy of the investigational drug over the placebo.

Moreover, as for the secondary efficacy endpoint, the efficacy and theside effects of the investigational drug were evaluated according to theinfluenza virus titer using a nasal or throat swab.

Patients who satisfied all of the following criteria were selected assubjects.

(a) Male or female patients at 12 years old or older and younger than 65years old

(b) Patients satisfying all of the following criteria and diagnosed withinfluenza virus infectious disease

Body temperature (axillary temperature) of 38.0° C. or higher

Having one or more moderate or severer symptoms among the followingsystemic symptoms and respiratory symptoms due to influenza virusinfectious disease

-   -   Systemic symptoms (headache, feverishness or chills, muscular or        joint pain, fatigue)    -   Respiratory symptoms (cough, sore throat, nasal congestion)        (c) Patients within 48 hours from onset (at registration)        The definition of onset is any of the following.

When the body temperature increased for the first, time (at least anincrease of 1° C. from normal temperature)

When any one or more of the systemic symptoms and respiratory symptomswere developed

Method for Administering Investigational Drug

(i) Test Drug

20 mg Tablet of Compound II-6

(ii) Placebo or Control Drug

Placebo for 20 mg tablet of Compound II-6

75 mg Capsule of Oseltamivir

Placebo for 75 mg capsule of Oseltamivir: Capsule undistinguishable from75 mg capsule of Oseltamivir

Dosage and Administration Method

Eligible patients at 20 to 64 years old were randomly allocated to agroup receiving a single administration of Compound II-6 (40 or 80 mgdepending on the body weight), a group receiving 75 mg Oseltamivir twicea day for 5 days, and a placebo group in a ratio of 2:2:1.

Eligible patients at 12 to 19 years old were randomly allocated to agroup receiving a single administration of Compound II-6 (40 or 80 mgdepending on the body weight) and a placebo administered group in aratio of 2-1.

The dosage of Compound II-6 was 40 mg for subjects weighing less than 80kg, and 80 mg for subjects weighing 80 kg or more.

Investigational Drug for Each Administered Group

[Compound II-6 Group]

Day 1:

20 mg Tablets of Compound II-6 were administered orally (2 tablets or 4tablets depending on the body weight). Placebo capsules for Oseltamivirwere administered orally twice a day (morning, evening), one capsule peradministration.

Day 2 to Day 5:

Placebo capsules for Oseltamivir were administered orally twice a day(morning, evening), one capsule per administration.

[Oseltamivir Group]

Day 1:

Placebo tablets for Compound II-6 were administered orally (2 tablets or4 tablets depending on the body weight). 75 mg Capsules of Oseltamivirwere administered orally twice a day (morning, evening), one capsule peradministration.

Day 2 to Day 5:

75 mg Capsules of Oseltamivir were administered orally twice a day(morning, evening), one capsule per administration.

[Placebo Group]

Day 1:

Placebo tablets for Compound II-6 were administered orally (2 tablets or4 tablets depending on the body weight). Placebo capsules forOseltamivir were administered orally twice a day (morning, evening), onecapsule per administration.

Day 2 to Day 5:

Placebo capsules for Oseltamivir were administered orally twice a day(morning, evening), one capsule per administration.

“Day 1” indicates the first day of administration, and “Day 2 to Day 5”indicates the second day to the fifth day as counted from the first dayof administration.

Main Efficacy Endpoint

The main efficacy endpoint is the time to alleviation of influenzasymptoms (the time to alleviation of influenza symptoms).

It is the time from the beginning of administration until alleviation ofinfluenza symptoms. Alleviation of influenza symptoms refers to when all7 influenza symptoms (cough, sore throat, headache, nasal congestion,feverishness or chills, muscular or joint pain, fatigue) become “0:none” or “1: mild” in the patient diary that the subject keeps, and thiscondition continues at least 21.5 hours (24 hours—10%).

Secondary Efficacy Endpoint

The secondary efficacy endpoint is as follows.

(1) Proportion of patients having a positive influenza virus titer ateach point

(2) Amount of change in virus titer from baseline at each point

(3) Time to termination of viral shedding based on virus titer

(4) Incidence of side effects

The virus Liter was measured in the following manner.

(1) MDCK-SIAT1 cells seeded in a flat-bottom 96-well microplate werecultured in a 5% CO₂ incubator at 37±1° C. for 1 day.

(2) A standard strain (influenza virus AII3N2, A/Victoria/361/2011,storage condition: −80° C., origin: National Institute of InfectiousDiseases), a sample (collected from patients in Phase III clinical testof Compound II-6 and stored in an ultra-low-temperature freezer), and amedium for cell control were diluted 101 to 107 folds by a 10-foldserial dilution method.(3) After cells present in a sheet form were confirmed under an invertedmicroscope, the medium was removed, and a new medium was added at 100μL/well.(4) The medium was removed.(5) Each of the samples (100 to 107) prepared at (2) above wasinoculated at 100 μL/well, using 4 wells per sample.(6) Centrifugal adsorption was performed at room temperature at 1000 rpmfor 30 minutes.(7) After centrifugation, the medium was removed, and cells were washedonce with a new medium.(8) A new medium was added at 100 μL/well.(9) Incubation was performed in a 5% CO₂ incubator at 33±1° C. for 3days.(10) After incubation, the CytoPathic Effect (CPE) was evaluated underan inverted microscope.Method for Determining to have a Positive Virus Titer

When the detection limit was exceeded as measured by the above virustiter measurement method, it was determined to be positive.

Analysis of Primary Endpoint

As for the time to alleviation of influenza symptoms, which is theprimary endpoint, the primary analysis and the secondary analysis aredescribed. The primary analysis was performed on the ITTI group.

(1) Primary Analysis

For patients at 12 to 64 years old, the placebo group and theinvestigational drug administered group were compared by stratifiedgeneralized Wilcoxon test using the total score of 7 influenza symptomsbefore administration (11 points or less, 12 points or more) and regions(Japan/Asia, other regions) as stratification factors.

Also, a Kaplan-Meier survival curve was drawn for each group tocalculate the median time to alleviation of influenza symptoms and the95% confidence interval thereof as well as the difference between thegroups in the time to alleviation of influenza symptoms and the 95%confidence interval thereof.

(2) Secondary Analysis

For patients at 20 to 64 years old, the time to alleviation of influenzasymptoms was compared between the Compound II-6 group and theOseltamivir group by the same method as the primary analysis.

Analysis of Secondary Endpoint

The following secondary efficacy endpoints were compared between theCompound II-6 group and the placebo group and between the Compound II-6group and the Oseltamivir group (the age group of 20 to 64 years old).

(1) Proportion of Patients Having a Positive Influenza Virus Titer atVarious Time Points

Only the patients having a virus titer equal to or greater than thedetermination limit before the beginning of administration in Visit 1were included in the analysis. In each Visit, a Mantel-Haenszel testusing the total score of 7 influenza symptoms before administration andthe regions as stratification factors was applied, and the proportion ofpatients having a positive virus titer was compared between two groups.

(2) Amount of Change in Virus Titer from Baseline at Various Time Points

Only the patients having a virus titer before the beginning ofadministration in Visit 1 were included in the analysis. In each Visit,a van Elteren test using the total score of 7 influenza symptoms beforeadministration and the regions as stratification factors was applied,and the amount of change in influenza virus titer from the baseline wascompared between two groups.

(3) Time to Termination of Viral Shedding Based on Virus Titer

Only the patients having a virus titer equal to or greater than thedetermination limit before the beginning of administration in Visit 1were included in the analysis. A stratified generalized Wilcoxon testusing the total score of 7 influenza symptoms before administration andthe regions as stratification factors was applied.

(4) Incidence of Side Effects

The number of side-effect episodes and the number of patients with sideeffect were counted for each administration group.

(1) Results of Primary Endpoint (Time to Alleviation of InfluenzaSymptoms)

Out of 1436 randomly selected patients, 1366 patients (578 patients inthe 40 mg or 80 mg Compound II-6 administered group, 498 patients in theOseltamivir administered group, and 290 patients in the placebo group)completed the test. As for the primary endpoint, the ITTI cases (caseswhere GCP was followed, the investigational drug was administered, andinfluenza virus infection was confirmed) consisted of 1064 patients.

The per protocol set cases consisted of 990 patients (427 patients inthe 40 mg or 80 mg Compound II-6 administered group, 351 patients in theOseltamivir administered group, and 212 patients in the placebo group).

Analysis results are shown in the following table.

TABLE 47 12 Years old or older and 20 Years old or older and youngerthan 65 years old younger than 65 years old Compound II-6 PlaceboCompound II-6 Oseltamivir administered group administered groupadministered group administered group Number of patients 455 230 375 377Median (hour) 53.7 80.2 53.5 53.8 95% Confidence 49.5, 58.5 72.6, 87.148.0, 58.5 50.2, 56.4 interval (hour) Difference between −26.5 — −0.3 —groups^(a) (hour) 95% Confidence −35.8, −17.8 — −6.6, 6.6 — interval ofdifference between groups (hour)^(b) Stratified generalized <.0001 —0.7560 — Wilcoxon test^(c) p Value^(a) ^(a)vs Placebo or vs Oseltamivir^(b)Bootstrap estimation ^(c)Used the regions and the total score of 7influenza symptoms before administration as stratification factors, andcensored at final evaluation for patients whose symptoms were noalleviated.

In the ITTI group, the time to alleviation of influenza symptoms(median) (95% CI) was 53.7 hours (95% CI: 49.5, 58.5) in the CompoundII-6 group while 80.2 hours (95% CI: 72.6, 87.1) in the placebo group,and the difference between the Compound II-6 group and the placebo groupwas −26.5 hours. The time to alleviation of influenza symptoms of theCompound II-6 group was significantly shorter than that of the placebogroup in the primary analysis using a stratified generalized Wilcoxontest (p<0.0001).

In the subgroup of patients at 20 years old or older and younger than 65years old, the time to alleviation of influenza symptoms was 53.5 hours(95% CI: 48.0, 58.5) in the Compound II-6 group while 53.8 hours (95%CI: 50.2, 56.4) in the Oseltamivir group, and the difference between theCompound II-6 group and the Oseltamivir group was −0.3 hours. There wasno significant difference between the times to alleviation of influenzasymptoms of the Compound II-6 group and the Oseltamivir group in thestratified generalized Wilcoxon test.

Analysis of Secondary Endpoint

(1) Proportion of Patients Having a Positive Influenza Virus Titer atVarious Points

Analysis results are shown in the following table.

TABLE 48 12 Years old or older and 20 Years old or older and youngerthan 65 years old younger than 65 years old Compound II-6 PlaceboCompound II-6 Oseltamivir administered administered administeredadministered ObservatIon group group group group time point N = 427 N =210 N = 352 N = 359 Day 2 Proportion 47.8% 96.0% 47.6% 91.0% (197/412) (193/201) (161/338) (315/346) 95% Confidence 42.9, 52.8 92.3, 98.3 42.2,53.1 87.5, 93.8 interval p Value ^(a) <.0001 — <.0001 — Day 3 Proportion21.5% 70.2% 19.8% 57.3% (87/404) (134/191)  (66/333) (197/344) 95%Confidence 17.6, 25.9 63.1, 76.5 15.7, 24.5 51.9, 62.6 Interval p Value^(a) <.0001 — <.0001 — Day 4 Proportion 16.7% 56.1% 16.1% 27.6% (19/114)(32/57) (14/87)  (29/105) 95% Confidence 10.3, 24.8 42.4, 69.3 9.1, 25.519.3, 37.2 interval p Value ^(a) <.0001 — 0.0852 — Day 5 Proportion13.6% 29.7% 13.0% 20.9% (55/403)  (57/192)  (43/331)  (70/335) 95%Confidence 10.4, 17.4 23.3, 36.7 9.6, 17.1 16.7, 25.6 interval p Value^(a) <.0001 — 0.0066 — Day 6 Proportion 8.2% 12.5% 5.6% 9.0% (8/97) (6/48)  (4/71)  (7/78) 95% Confidence 3.6, 15.6 4.7, 25.2 1.6, 13.83.7, 17.6 interval p Value ^(a) 0.4767 — 0.6187 — Day 9 Proportion 2.9%4.6% 3.0% 3.2% (12/407)   (9/197)  (10/335)  (11/339) 95% Confidence1.5, 5.1 2.1, 8.5 1.4, 5.4 1.6, 5.7 interval p Value ^(a) 0.3379  0.8618 —Day 2 indicates 24 hours later, as counted from the first day ofadministration, Day 3 indicates 48 hours later, Day 4 indicates 72 hourslater, Day 5 indicates 96 hours later, Day 6 indicates 120 hours later,and Day 9 indicates 192 hours later,a vs Placebo or vs Oseltamivir. Mantel-Haenszel test. Used the regionsand the total score of 7 influenza symptoms before administration asstratification factors, and intended for a group having a positive virusLiter before administration.

The proportion of patients having a positive virus titer wassignificantly lower in the Compound II-6 group than in the placebo groupon Day 2 (Mantel-Haenszel test: p≤0.0001), and likewise, significantlylower in the Compound II-6 group than in the placebo group on Day 3(p<0.0001). In the subgroup of patients at 20 years old or older andyounger than 65 years old, the proportion of patients having a positivevirus titer was significantly lower in the Compound II-6 group than inthe Oseltamivir group on Day 2 and Day 3 (p<0.0001).

(2) Amount of Change in Virus Titer from Baseline at Various Points

Analysis results are shown in the following table.

TABLE 49 12 Years old or older and 20 Years old or older and youngerthan 65 years old younger than 65 years old Compound II-6 PlaceboCompound II-6 Oseltamivir administered administered administeredadministered Observation group group group group time point N = 427 N =210 N = 352 N = 359 Day 2 Number of patients 412 201 338 346 Mean −4.44−1.19 −4.39 −2.51 Standard deviation 2.03 2.43 2.07 2.03 p Value ^(a)<.0001 — <.0001 — Day 3 Number of patients 404 191 333 344 Mean −4.82−2.91 −4.78 −4.20 Standard deviation 1.99 2.85 2.03 2.02 p Value ^(a)<.0001 — <.0001 — Day 4 Number of patients 114 57 87 105 Mean −4.50−3.31 −4.46 −4.63 Standard deviation 2.02 2.34 2.03 1.89 p Value ^(a)0.0008 — 0.8010 — Day 5 Number of patients 403 192 331 335 Mean −4.95−4.47 −4.95 −4.98 Standard deviation 1.93 2.21 1.94 1.82 p Value ^(a)0.0132 — 0.9425 — Day 6 Number of patients 97 48 71 78 Mean −4.58 −4.68−4.56 −4.85 Standard deviation 1.99 2.12 1.99 1.95 p Value ^(a) 0.9307 —0.2256 — Day 9 Number of patients 407 197 335 339 Mean −5.06 −4.87 −5.03−5.22 Standard deviation 1.87 1.85 1.89 1.70 p Value ^(a) 0.1684 —0.3267 —Unit: log₁₀ [TCID₅₀/mL].Day 2 indicates 24 hours later, as counted from the first day ofadministration, Day 3 indicates 48 hours later, Day 4 indicates 72 hourslater, Day 5 indicates 96 hours later, Day 6 indicates 120 hours later,and Day 9 indicates 192 hours later,a vs Placebo or vs Oseltamivir, van Elteren test. Used the regions andthe total score of 7 influenza symptoms before administration asstratification factors. Intended for a group having a positive virustiter before administration.

The virus titer decreased significantly in the Compound II-6 group ascompared to the placebo group on Day 2, and likewise, decreasedsignificantly as compared to the placebo group on Day 3 (van Elterentest: p<0.0001). In the subgroup of patients at 20 years old or olderand younger than 65 years old, the virus titer decreased significantlyin the Compound II-6 group as compared to the Oseltamivir group on Day 2and Day 3 (p<0.0001).

(3) Time to Termination of Viral Shedding Based on Virus Titer

Analysis results are shown in the following table.

TABLE 50 12 Years old or older and 20 Years old or older and youngerthan 65 years old younger than 65 years old Compound II-6 PlaceboCompound II-6 Oseltamivir administered administered administeredadministered group group group group Number of patients 423 207 348 35595% Confidence interval 24.0, 48.0 — 24.0, 48.0 72.0, 96.0 (hour)Difference between −72.0 — −48.0 — groups (hour) ^(a) Stratifiedgeneralized <.0001 — <.0001 — Wilcoxon test ^(b) p Value ^(a) vs Placeboor vs Oseltamivir. ^(b) Used the regions and the total score of 7influenza symptoms before administration as stratification factors.Censored at final evaluation for patients whose virus titer was noteliminated. Intended for analyzing patients who had a positive virustiter on Day 1 and whose data concerning the time to termination ofviral shedding was not missing.

The time (median) to termination of viral shedding based on virus titerwas 24.0 hours in the Compound II-6 group while 96.0 hours in theplacebo group, and was significantly shorter in the Compound II-6 groupthan in the placebo group (stratified generalized Wilcoxon test:p≤0.0001). The time to termination of viral shedding in the subgroup ofpatients at 20 years old or older and younger than 65 years old was 24.0hours in the Compound II-6 group and 72.0 hours in the Oseltamivirgroup, and was significantly shorter in the Compound II-6 group than inthe Oseltamivir group (p<0.0001).

(4) Incidence of Adverse Events

Severe adverse events the causal relationship of which cannot be deniedare not reported. Adverse events the causal relationship of which cannotbe denied occurred in 27 patients out of 610 patients (4.4%, 37episodes) in the Compound II-6 group, 12 patients out of 309 patients(3.9%, 19 episodes) in the placebo group, and 43 patients out of 513patients (8.4%, 53 episodes) in the Oseltamivir group. There was nostatistically significant difference between the incidences in theCompound II-6 group and the placebo group (Fisher's exact test,two-sided P value: 0.8627). However, the incidence in the Compound II-6group was significantly lower than that in the Oseltamivir group(Fisher's exact test, two-sided P value: 0.0088).

Test Example 17: Clinical Test (Ph3: Child)

The efficacy and safety of a single oral administration of aninvestigational drug (active ingredient (Compound II-6): 5 mg, 10 mg, 20mg, 40 mg) to patients infected by influenza virus were evaluated. Asfor the primary endpoint, guardians or subjects by themselves madeevaluations and measurements concerning the time to alleviation ofinfluenza symptoms (the time from the beginning of administration of theinvestigational drug until influenza symptoms (“cough”, “runnynose/nasal congestion”, and “fever”) were alleviated) to evaluate theefficacy of the investigational drug.

“Cough” and “runny nose/nasal congestion” were evaluated on a 4-pointscale [0: none, 1: mild, 2: moderate, 3: severe].

Patients who satisfied all of the following criteria were selected assubjects.

(a) Male or female patients at 6 months old or older and younger than 12years old

(b) Patients satisfying all of the following criteria and diagnosed withinfluenza virus infectious disease

Positive in influenza rapid diagnosis [Rapid antigen test (RAT)] basedon a nasal or throat swab

Body temperature (axillary temperature) of 38.0° C. or higher

Having one or more moderate or severer symptoms among the respiratorysymptoms due to influenza virus infectious disease for patients at 7years old or older

(c) Patients within 48 hours from onset (at registration). The onset isdefined as when the body temperature exceeding 37.5° C. is confirmed forthe first time.

(d) Patients having a body weight of 5 kg or more.

Method for Administering Investigational Drug

(i) Test Drug

5 mg Tablet of Compound II-6: Half of 10 mg tablet of Compound II-6

10 mg Tablet of Compound II-6

20 mg Tablet of Compound II-6

Dosage and Administration Method

Patients received a single oral administration on Day 1 in a dosecalculated based on the body weight (see the table below).

TABLE 51 Body weight of patient at the Dose of Compound time ofscreening Compound II-6 II-6 tablet 5 kg or more and less  5 mg Half of10 mg tablet than 10 kg 10 kg or more and less 10 mg One 10 mg tabletthan 20 kg 20 kg or more and less 20 mg One 20 mg tablet than 40 kg ortwo 10 mg tablets 40 kg or more 40 mg Two 20 mg tabletsMain Efficacy Endpoint

The main efficacy endpoint is the time to alleviation of influenzasymptoms (the time to alleviation of influenza symptoms).

It is the time from the beginning of administration until alleviation ofinfluenza symptoms. Alleviation of an influenza symptom refers to thetime when a and b below are satisfied from the beginning ofadministration, and this clinical condition continues at least 21.5hours (24 hours—10%).

a. “Cough” and “runny nose/nasal congestion” are both “0: none” or “1:mild” in the patient diary

b. Body temperature (axillary temperature) is lower than 37.5° C.

Analysis of Primary Endpoint

As for the time to alleviation of influenza symptoms, which is theprimary endpoint, the primary analysis is described. The primaryanalysis was performed on the ITTI group.

(1) Primary Analysis

A Kaplan-Meier curve of the time to alleviation of influenza symptoms(“cough”, “runny nose/nasal congestion”, and “fever”) (the time toalleviation of influenza symptoms) was drawn to calculate the mediantime to complete alleviation of influenza symptoms and the 95%confidence interval thereof. Patients whose influenza symptoms were notcompletely alleviated during the observation period were treated ascensored cases.

(1) Results of Primary Endpoint (Time to Alleviation of InfluenzaSymptoms)

As for the primary endpoint, 103 patients were involved. The time(median) to alleviation of influenza symptoms in the ITTI group was 44.6hours (95% CI: 38.9, 62.5).

Formulation Example

The following Formulation Examples are only exemplified and not intendedto limit the scope of the invention.

Formulation Example 1: Tablets

The compounds used in the present invention, lactose and calciumstearate are mixed. The mixture is crushed, granulated and dried to givea suitable size of granules. Next, calcium stearate is added to thegranules, and the mixture is compressed and molded to give tablets.

Formulation Example 2: Capsules

The compounds used in the present invention, lactose and calciumstearate are mixed uniformly to obtain powder medicines in the form ofpowders or fine granules. The powder medicines are filled into capsulecontainers to give capsules.

Formulation Example 3: Granules

The compounds used in the present invention, lactose and calciumstearate are mixed uniformly and the mixture is compressed and molded.Then, it is crushed, granulated and sieved to give suitable sizes ofgranules.

Formulation Example 4: Orally Disintegrated Tablets

The compounds used in the present invention and crystalline celluloseare mixed, granulated and tablets are made to give orally disintegratedtablets.

Formulation Example 5: Dry Syrups

The compounds used in the present invention and lactose are mixed,crushed, granulated and sieved to give suitable sizes of dry syrups.

Formulation Example 6: Injections

The compounds used in the present invention and phosphate buffer aremixed to give injection.

Formulation Example 7: Infusions

The compounds used in the present invention and phosphate buffer aremixed to give injection.

Formulation Example 8: Inhalations

The compound used in the present invention and lactose are mixed andcrushed finely to give inhalations.

Formulation Example 9: Ointments

The compounds used in the present invention and petrolatum are mixed togive ointments.

Formulation Example 10: Patches

The compounds used in the present invention and base such as adhesiveplaster or the like are mixed to give patches.

INDUSTRIAL APPLICABILITY

The parent compounds used in the present invention have cap-dependentendonuclease (CEN) inhibitory activity after absorption into the body.The compounds (the parent compounds and/or the prodrugs) used in thepresent invention can be useful agents for treatment and/or preventionof symptom and/or disease induced by infection with influenza virus. Thepharmaceutical composition is effective for shortening the time toalleviation of influenza symptoms, and is useful for treating and/orpreventing an influenza virus infectious disease.

The invention claimed is:
 1. A method for treating a disease caused by avirus having cap-dependent endonuclease comprising administering to apatient in need thereof a crystal of the compound of the followingformula:

wherein the crystal is selected from the group consisting of: (i)crystal Form I having two or more peaks in diffraction angles (2θ)selected from 8.6±0.2°, 14.1±0.2°, 17.4±0.2°, 20.0±0.2°, 24.0±0.2°,26.3±0.2°, 29.6±0.2° and 35.4±0.2° in an X-ray powder diffractionspectrum, (ii) crystal Form II having two or more peaks in diffractionangles (2θ) selected from 4.4±0.2°, 8.9±0.2°, 11.7±0.2°, 14.9±0.2°,22.3±0.2°, 24.4±0.2°, 28.0±0.2° and 31.5±0.2° in an X-ray powderdiffraction spectrum, and (iii) crystal Form III having two or morepeaks in diffraction angles (2θ) selected from 4.4±0.2°, 8.7±0.2°,10.6±0.2°, 17.3±0.2°, 17.5±0.2°, 22.0±0.2°, 24.0±0.2°, 24.1±0.2° and31.0±0.2° in an X-ray powder diffraction spectrum.
 2. The methodaccording to claim 1, wherein the crystal is (i) crystal Form I havingpeaks in diffraction angles (2θ) of 8.6±0.2°, 14.1±0.2°, 17.4±0.2°,20.0±0.2°, 24.0±0.2°, 26.3±0.2°, 29.6±0.2° and 35.4±0.2° in an X-raypowder diffraction spectrum.
 3. The method according to claim 1, whereinthe (i) crystal Form I is administered orally in a single dose.
 4. Themethod according to claim 1, wherein 40 mg or 80 mg of the (i) crystalForm I is administered in a single dose.
 5. The method according toclaim 1, wherein 5 to 40 mg of the (i) crystal Form I is administered ina single dose depending on the body weight of the patient.
 6. The methodaccording to claim 1, wherein 40 mg of the (i) crystal Form I isadministered in two tablets of 20 mg per tablet.
 7. The method accordingto claim 1, wherein the (i) crystal Form I is administered and thepatient is a human 12 years old or older.
 8. The method according toclaim 1, wherein the crystal is (ii) crystal Form II having peaks indiffraction angles (2θ) of 4.4±0.2°, 8.9±0.2°, 11.7±0.2°, 14.9±0.2°,22.3±0.2°, 24.4±0.2°, 28.0±0.2° and 31.5±0.2° in an X-ray powderdiffraction spectrum.
 9. The method according to claim 1, wherein the(ii) crystal Form II is administered orally in a single dose.
 10. Themethod according to claim 1, wherein 40 mg or 80 mg of the (ii) crystalForm II is administered in a single dose.
 11. The method according toclaim 1, wherein 5 to 40 mg of the (ii) crystal Form II is administeredin a single dose depending on the body weight of the patient.
 12. Themethod according to claim 1, wherein 40 mg of the (ii) crystal Form IIis administered in two tablets of 20 mg per tablet.
 13. The methodaccording to claim 1, wherein the (ii) crystal Form II is administeredand the patient is a human 12 years old or older.
 14. The methodaccording to claim 1, wherein the crystal is (iii) crystal Form IIIhaving peaks in diffraction angles (2θ) of 4.4±0.2°, 8.7±0.2°,10.6±0.2°, 17.3±0.2°, 17.5±0.2°, 22.0±0.2°, 24.0±0.2°, 24.1±0.2° and31.0±0.2° in an X-ray powder diffraction spectrum.
 15. The methodaccording to claim 1, wherein the (iii) crystal Form III is administeredorally in a single dose.
 16. The method according to claim 1, wherein 40mg or 80 mg of the (iii) crystal Form III is administered in a singledose.
 17. The method according to claim 1, wherein 5 to 40 mg of the(iii) crystal Form III is administered in a single dose depending on thebody weight of the patient.
 18. The method according to claim 1, wherein40 mg of the (iii) crystal Form III is administered in two tablets of 20mg per tablet.
 19. The method according to claim 1, wherein the (iii)crystal Form III is administered and the patient is a human 12 years oldor older.
 20. A method for treating influenza comprising administeringto a patient in need thereof a crystal of the compound of the followingformula:

wherein the crystal is selected from the group consisting of: (i)crystal Form I having two or more peaks in diffraction angles (2θ)selected from 8.6±0.2°, 14.1±0.2°, 17.4±0.2°, 20.0±0.2°, 24.0±0.2°,26.3±0.2°, 29.6±0.2° and 35.4±0.2° in an X-ray powder diffractionspectrum, (ii) crystal Form II having two or more peaks in diffractionangles (2θ) selected from 4.4±0.2°, 8.9±0.2°, 11.7±0.2°, 14.9±0.2°,22.3±0.2°, 24.4±0.2°, 28.0±0.2° and 31.5±0.2° in an X-ray powderdiffraction spectrum, and (iii) crystal Form III having two or morepeaks in diffraction angles (2θ) selected from 4.4±0.2°, 8.7±0.2°,10.6±0.2°, 17.3±0.2°, 17.5±0.2°, 22.0±0.2°, 24.0±0.2°, 24.1±0.2° and31.0±0.2° in an X-ray powder diffraction spectrum.
 21. The methodaccording to claim 20, wherein the crystal is (i) crystal Form I havingpeaks in diffraction angles (2θ) of 8.6±0.2°, 14.1±0.2°, 17.4±0.2°,20.0±0.2°, 24.0±0.2°, 26.3±0.2°, 29.6±0.2° and 35.4±0.2° in an X-raypowder diffraction spectrum.
 22. The method according to claim 20,wherein the (i) crystal Form I is administered orally in a single dose.23. The method according to claim 20, wherein 40 mg or 80 mg of the (i)crystal Form I is administered in a single dose.
 24. The methodaccording to claim 20, wherein 5 to 40 mg of the (i) crystal Form I isadministered in a single dose depending on the body weight of thepatient.
 25. The method according to claim 20, wherein 40 mg of the (i)crystal Form I is administered in two tablets of 20 mg per tablet. 26.The method according to claim 20, wherein the (i) crystal Form I isadministered and the patient is a human 12 years old or older.
 27. Themethod according to claim 20, wherein the crystal is (ii) crystal FormII having peaks in diffraction angles (2θ) of 4.4±0.2°, 8.9±0.2°,11.7±0.2°, 14.9±0.2°, 22.3±0.2°, 24.4±0.2°, 28.0±0.2° and 31.5±0.2° inan X-ray powder diffraction spectrum.
 28. The method according to claim20, wherein the (ii) crystal Form II is administered orally in a singledose.
 29. The method according to claim 20, wherein 40 mg or 80 mg ofthe (ii) crystal Form II is administered in a single dose.
 30. Themethod according to claim 20, wherein 5 to 40 mg of the (ii) crystalForm II is administered in a single dose depending on the body weight ofthe patient.
 31. The method according to claim 20, wherein 40 mg of the(ii) crystal Form II is administered in two tablets of 20 mg per tablet.32. The method according to claim 20, wherein the (ii) crystal Form IIis administered and the patient is a human 12 years old or older. 33.The method according to claim 20, wherein the crystal is (iii) crystalForm III having peaks in diffraction angles (2θ) of 4.4±0.2°, 8.7±0.2°,10.6±0.2°, 17.3±0.2°, 17.5±0.2°, 22.0±0.2°, 24.0±0.2°, 24.1±0.2° and31.0±0.2° in an X-ray powder diffraction spectrum.
 34. The methodaccording to claim 20, wherein the (iii) crystal Form III isadministered orally in a single dose.
 35. The method according to claim20, wherein 40 mg or 80 mg of the (iii) crystal Form III is administeredin a single dose.
 36. The method according to claim 20, wherein 5 to 40mg of the (iii) crystal Form III is administered in a single dosedepending on the body weight of the patient.
 37. The method according toclaim 20, wherein 40 mg of the (iii) crystal Form III is administered intwo tablets of 20 mg per tablet.
 38. The method according to claim 20,wherein the (iii) crystal Form III is administered and the patient is ahuman 12 years old or older.
 39. A method for preventing influenzacomprising administering to a patient in need thereof a crystal of thecompound of the following formula:

wherein the crystal is selected from the group consisting of: (i)crystal Form I having two or more peaks in diffraction angles (2θ)selected from 8.6±0.2°, 14.1±0.2°, 17.4±0.2°, 20.0±0.2°, 24.0±0.2°,26.3±0.2°, 29.6±0.2° and 35.4±0.2° in an X-ray powder diffractionspectrum, (ii) crystal Form II having two or more peaks in diffractionangles (2θ) selected from 4.4±0.2°, 8.9±0.2°, 11.7±0.2°, 14.9±0.2°,22.3±0.2°, 24.4±0.2°, 28.0±0.2° and 31.5±0.2° in an X-ray powderdiffraction spectrum, and (iii) crystal Form III having two or morepeaks in diffraction angles (2θ) selected from 4.4±0.2°, 8.7±0.2°,10.6±0.2°, 17.3±0.2°, 17.5±0.2°, 22.0±0.2°, 24.0±0.2°, 24.1±0.2° and31.0±0.2° in an X-ray powder diffraction spectrum.
 40. The methodaccording to claim 39, wherein the crystal is (i) crystal Form I havingpeaks in diffraction angles (2θ) of 8.6±0.2°, 14.1±0.2°, 17.4±0.2°,20.0±0.2°, 24.0±0.2°, 26.3±0.2°, 29.6±0.2° and 35.4±0.2° in an X-raypowder diffraction spectrum.
 41. The method according to claim 39,wherein the (i) crystal Form I is administered orally in a single dose.42. The method according to claim 39, wherein 40 mg or 80 mg of the (i)crystal Form I is administered in a single dose.
 43. The methodaccording to claim 39, wherein 5 to 40 mg of the (i) crystal Form I isadministered in a single dose depending on the body weight of thepatient.
 44. The method according to claim 39, wherein 40 mg of the (i)crystal Form I is administered in two tablets of 20 mg per tablet. 45.The method according to claim 39, wherein the (i) crystal Form I isadministered and the patient is a human 12 years old or older.
 46. Themethod according to claim 39, wherein the crystal is (ii) crystal FormII having peaks in diffraction angles (2θ) of 4.4±0.2°, 8.9±0.2°,11.7±0.2°, 14.9±0.2°, 22.3±0.2°, 24.4±0.2°, 28.0±0.2° and 31.5±0.2° inan X-ray powder diffraction spectrum.
 47. The method according to claim39, wherein the (ii) crystal Form II is administered orally in a singledose.
 48. The method according to claim 39, wherein 40 mg or 80 mg ofthe (ii) crystal Form II is administered in a single dose.
 49. Themethod according to claim 39, wherein 5 to 40 mg of the (ii) crystalForm II is administered in a single dose depending on the body weight ofthe patient.
 50. The method according to claim 39, wherein 40 mg of the(ii) crystal Form II is administered in two tablets of 20 mg per tablet.51. The method according to claim 39, wherein the (ii) crystal Form IIis administered and the patient is a human 12 years old or older. 52.The method according to claim 39, wherein the crystal is (iii) crystalForm III having peaks in diffraction angles (2θ) of 4.4±0.2°, 8.7±0.2°,10.6±0.2°, 17.3±0.2°, 17.5±0.2°, 22.0±0.2°, 24.0±0.2°, 24.1±0.2° and31.0±0.2° in an X-ray powder diffraction spectrum.
 53. The methodaccording to claim 39, wherein the (iii) crystal Form III isadministered orally in a single dose.
 54. The method according to claim39, wherein 40 mg or 80 mg of the (iii) crystal Form III is administeredin a single dose.
 55. The method according to claim 39, wherein 5 to 40mg of the (iii) crystal Form III is administered in a single dosedepending on the body weight of the patient.
 56. The method according toclaim 39, wherein 40 mg of the (iii) crystal Form III is administered intwo tablets of 20 mg per tablet.
 57. The method according to claim 39,wherein the (iii) crystal Form III is administered and the patient is ahuman 12 years old or older.